Pyrimidine derivatives and oligonucleotides containing same

ABSTRACT

Compounds having structure (1) wherein R1 is -H a protecting group, a linker or a binding partner; and R2 and R34 are as defined in the specification. The invention also provides intermediates and methods make the structure (1) compounds, as well as methods to use the compounds as labels in diagnostic assays and to enhance binding to complementary bases.

BACKGROUND OF THE INVENTION

This invention relates to the field of labels, particularly labels fordiagnostic or analytical use. In particular, it relates tooligonucleotides that are modified to enhance the binding affinity orthe binding specificity of the oligonucleotides for complementarysequences and that in addition optionally bear a readily detectablecharacteristic.

Sequence specific binding of oligonucleotides both to single strandedRNA and DNA and to duplex DNA is widely known. This phenomenon has beenharnessed for a great variety of diagnostic, therapeutic and analytical,e.g., sequence determination or gene mapping, purposes. Previously, oneobjective of research in this field has been to increase the affinity ofsuch oligonucleotides for their complementary sequences. For example,workers have described oligonucleotides containing 5-substitutedpyrimidine bases that substantially increase the Tm for oligonucleotidebinding to complementary bases (International Publication No. WO93/10820).

Publications have described the use of fluorescent cytosine derivativesto prepare labeled DNA probes. See Inoue et al., Jpn. Kokai JP 62059293,(1987). In addition, fluorescent labeled nucleotides have been employedin DNA sequencing. See Prober et al., "Science" 238:336-341 (1987).

1,3-Dihydro-2H-imidazo[4,5-b]-quinolin-2-one derivatives asphosphodiesterase inhibitors are disclosed by Raeymaekers et al. (EP541,153).

U.S. Pat. No. 5,502,177, discloses phenoxazine polycycle-containingoligonucleotides and monomers for preparing the oligonucleotides.

OBJECTS OF THE INVENTION

The invention compositions or methods accomplish one or more of thefollowing objects.

An object of this invention is to increase the affinity ofoligonucleotides for their complementary sequences.

An object of this invention is to increase the specificity ofoligonucleotides for their complementary sequences.

Another object of this invention is to provide detectable labels for usein diagnostic assays.

Another object is to enhance diagnostic assays that useoligonucleotides.

Another object is to improve the therapeutic efficacy ofoligonucleotides.

Another object is to improve the potency of oligonucleotides asantisense reagents that affect gene expression by altering intracellularmetabolism of complementary RNA sequences encoding a target gene(s).

Another object is to provide chemical intermediates and synthesismethods to prepare the invention compositions.

These and other objects of the invention will be apparent when oneconsiders the disclosure as a whole.

SUMMARY OF THE INVENTION

In accordance with the objects, the invention provides compounds havingthe structure (1) ##STR2## and tautomers, solvates and salts thereof,wherein R¹ is a binding partner, a protecting group, a linker or --H;

R² is A(Z)_(X1), wherein A is a spacer and Z independently is a labelbonding group optionally bonded to a detectable label, but R² is notamine, protected amine, nitro or cyano;

R²⁷ is independently --CH═, --N═, --C(C₁₋₈ alkyl)═ or --C(halogen)═, butno adjacent R²⁷ are both --N═, or two adjacent R²⁷ are taken together toform a ring having the structure, ##STR3## where R^(a) is independently--CH═, --N═, --C(C₁₋₈ alkyl)═ or --C(halogen)═, but no adjacent R^(a)are both --N═;

R³⁴ is --O--, --S-- or --N(CH₃)--; and

X1 is 1, 2 or 3.

When the binding partner R¹ is an oligonucleotide, embodiments of thecompounds of this invention include oligonucleotides of structure (2),(2A), (2B), or (2C) ##STR4## wherein D is --OH, protected --OH, anoligonucleotide coupling group or a solid support;

D¹ is an oligonucleotide coupling group, --OH, protected --OH or a solidsupport, wherein D¹ is bonded to one 2' or 3' position in theoligonucleotide of structure (2) and the adjacent 2' or 3' position instructure (2) is substituted with R²¹, provided that D and D¹ are notboth an oligonucleotide coupling group or they are not both a solidsupport;

D² is --CO₂ R⁵, --C(O)N(R⁵)₂, --SO₃ R⁵, --SO₂ N(R⁵)₂ or an activatedderivative of --CO₂ H or --SO₃ H;

D³ is a protecting group, --H or --(CH₂)₂₋₆ --N(R⁵)₂ ;

R⁴ is independently a phosphodiester linkage or a phosphodiestersubstitute linkage, wherein R⁴ is bonded to one 2' or 3' position in thestructure (2) oligonucleotide and the adjacent 2' or 3' position instructure (2) is substituted with R²¹ ;

R⁵ is independently --H or a protecting group;

R²¹ is independently --H, --OH, halogen or a moiety that enhances theoligonucleotide against nuclease cleavage;

R³⁷ is independently --O--, --CH₂ -- or --CF₂ --;

n is an integer from 0 to 98; and

B independently is a purine or pyrimidine base or a protected derivativethereof, provided that at least one B is a base of structure (3)##STR5##

Embodiments include compositions useful as intermediates in making thestructure (1) compounds, including intermediates having structure (4)##STR6## and tautomers, solvates and salts thereof wherein, R²⁴ is ahalogen; and

R²⁵ is --SH, --OH, ═S or ═O.

In a further embodiment, the invention includes contacting a structure(2), (2A), (2B), or (2C) oligonucleotide, wherein n is at least about 7,with a sample suspected to contain a nucleic acid having a base sequencethat is at least substantially complementary to the structure (2), (2A),(2B), or (2C) oligonucleotide.

In a further embodiment, the invention includes detecting the presence,absence or amount of a complex comprising a structure (2), (2A), (2B),or (2C) oligonucleotide, wherein n is at least about 7, and a nucleicacid having a base sequence that is at least substantially complementaryto the structure (2), (2A), (2B), or (2C) oligonucleotide.

In a further embodiment, the invention includes converting a structure(4) compound to a compound of structure (1) where R³⁴ is --O-- or --S--and the R² atom or moiety alpha to the ring containing R²⁷ is --O--,--S-- or --CH₂ --, by displacing R²⁴.

In a further embodiment, the invention includes converting a structure(4A) compound (a structure (1) compound where R² is replaced with--NH₂); to a compound of structure (1), by reductive alkylation of the--NH₂ group to yield a structure (1) compound where the R² moiety alphato the ring containing R²⁷ is --NH--.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises all combinations formed by independentlyselecting individual Markush group members and assembling them inaccordance with the teachings herein. The invention optionally excludesany feature or substance found in, or obvious over, the prior art.

Structural formulas are designated as parenthetical numerals. We intendthat designation of aromaticity with respect to carbocycles andheterocycles herein includes any highly resonant unsaturated ringstructure. Alternatively, placement of double bonds, where indicated,represents one potential structure for the depicted compound but weintend this depiction to include other resonant states of the compoundas well as protonated and charged species, only one of which a structuremay show.

The invention includes invention compounds in unpurified, substantiallypurified and purified forms. It includes invention compounds that arepresent with any additional component(s) such as a solvent, reactant orby-product that is present during invention compound synthesis orpurification, and any additional component(s) that is present during theuse or manufacture of an invention compound.

Halo and halogen mean F, Cl, Br or I.

Alkyl means unbranched, branched or cyclic hydrocarbons that aresaturated or unsaturated, or combinations thereof. Alkyl includes allisomers, e.g., stereoisomers, positional isomers, diastereomers andregioisomers. Alkyl moieties that are unsaturated will typically contain1, 2, 3 or more --CH═CH-- or --C.tbd.C-- groups, usually one such group.

Substituted alkyl means unbranched, branched or cyclic hydrocarbons thatare saturated or unsaturated, or combinations thereof, where thehydrocarbon contains a heteroatom linked to a carbon or a heteroatomthat replaces a carbon atom. Substituted alkyl includes all isomers,e.g., stereoisomers, positional isomers, diastereomers and regioisomers.Substituted alkyl moieties that are unsaturated will typically contain1, 2, 3 or more --CH═CH-- or --C.tbd.--C-- groups, usually one suchgroup. Substituted alkyl includes alkyl groups having substituentslinked to a carbon atom or substituents that interrupt a carbon atomchain, and unless otherwise defined, substituents include ethers(--O--), ketones (--C(O)--), --OR⁵, --C(O)OR⁵, --C(O)O--, --OC(O)--,--C(O)H, --OCH₂ --, --OCH₂ CH₂ --, --OCH₂ O--, --OCH₂ CH₂ O--, --NR⁵ --,--NHR⁵, --NHC(O)--, --C(O)NH--, C(O)NHR⁵, --OC(O)NR⁵ --, --OC(O)NHR⁵,--NR⁵ C(O)NR⁵ --, --NR⁵ C(O)NHR⁵, --NR⁵ CH₂ --, --NR⁵ CH₂ CH₂ --, --S--,--SR⁵, --S(O)--, --S(O)(O)--, --S(O)OR⁵, --S(O)H, halogen, CN, NO₂, andcombinations of these substituents where R⁵ is hydrogen or a protectinggroup.

The invention compounds herein do not include obviously unstablestructures, e.g., --O--O--, --O--S-- or unsaturated cyclopropyl, unlessthey are useful as transitory intermediates in the preparation of morestable compounds.

As used herein, "monosaccharide" means a polyhydroxy aldehyde or ketonehaving the empirical formula (CH₂ O)_(n) where n is 3, 4, 5, 6 or 7.Monosaccharide includes open-chain and closed-chain forms, but willusually be closed chain forms. Monosaccharide includes hexofuranose andpentofuranose sugars such as 2'-deoxyribose, ribose, arabinose, xylose,their 2'-deoxy and 3'-deoxy derivatives, their 2',3'-dideoxyderivatives, and their derivatives containing R²¹ linked to the 2' or 3'position, usually the 2' position. Monosaccharide also includes the2',3' dideoxydidehydro derivative of ribose. Monosaccharides include theD- and L-isomers of glucose, fructose, mannose, idose, galactose,allose, gulose, altrose, talose, fucose, erythrose, threose, lyxose,erythrulose, ribulose, xylulose, ribose, arabinose, xylose, psicose,sorbose, tagatose, glyceraldehyde, dihydroxyacetone and their monodeoxyderivatives such as rhamnose. Monosaccharides are optionally protectedor partially protected.

As used herein, a "protecting group" means a moiety that prevents theatom to which it is linked from participating in unwanted reactions. Forexample, for --OR⁵, R⁵ is a protecting group for the oxygen atom foundin a hydroxyl or carboxyl group, for --SR⁵, R⁵ is a protecting group forsulfur in thiols for instance, and for --NHR⁵ or --N(R⁵)--, R⁵ is anitrogen atom protecting group for primary or secondary amines.Hydroxyl, amine and other reactive groups are found in inventioncompounds at, e.g., R², R²¹ or oligonucleotide linkages oroligonucleotide bases. These groups may require protection againstreactions taking place elsewhere in the molecule. The protecting groupsfor oxygen, sulfur or nitrogen atoms are usually used to preventunwanted reactions with electrophilic compounds, such as acylating orphosphorylating agents used, e.g., in nucleoside, nucleotide oroligonucleotide chemistry.

Protecting groups are intended to be removed by known procedures,although it will be understood that the protected intermediates fallwithin the scope of this invention. The removal of the protecting groupmay be arduous or straight-forward, depending upon the economics andnature of the conversions involved. In general, one will use aprotecting group with exocyclic amines in the B groups of the compoundsof this invention. For oligonucleotide containing such B groups to befully binding competent, exocyclic amines must be deprotected becausethe amine groups participate in hydrogen bonding with complementarybases. Similarly, one will typically use reversible protecting groupsfor the 5' and 3' hydroxyl groups of pentofuranose sugars in nucleotidesintended for use as monomers in synthesis of oligonucleotides containing3,5' linkages. Protecting groups commonly are employed to protectagainst covalent modification of a sensitive group in reactions such asphosphorylation, alkylation or acylation. Ordinarily, protecting groupsare removed by, e.g. hydrolysis, elimination or aminolysis. Thus, simplefunctional considerations will suffice to guide the selection of areversible or an irreversible protecting group at a given locus on theinvention compounds. Suitable protecting groups and criteria for theirselection are described in T. W. Greene and P. G. M. Wuts, Eds."Protective Groups in Organic Synthesis" 2nd edition, Wiley Press, atpps. 10-142, 143-174, 175-223, 224-276, 277-308, 309-405 and 406-454.

Salts

Embodiments include salts and complexes of invention compounds,including pharmaceutically acceptable or salts that are relativelynon-toxic. The invention compounds may have one or more moieties thatcarry at least a partial positive or negative charge in aqueoussolutions, typically at a pH of about 4-10, that can participate informing a salt, a complex, a composition with partial salt and partialcomplex properties or other noncovalent interactions, all of which werefer to as a "salt(s)". Salts are usually biologically compatible orpharmaceutically acceptable or non-toxic, particularly for mammaliancells. Salts that are biologically toxic are optionally used withsynthetic intermediates of invention compounds. When a water solublecomposition is desired, monovalent salts are usually preferred.

Metal salts typically are prepared by reacting the metal hydroxide witha compound of this invention. Examples of metal salts which areoptionally prepared in this way are salts containing Li⁺, Na⁺, and K⁺. Aless soluble metal salt can be precipitated from the solution of a moresoluble salt by adding a suitable metal compound. Invention salts may beformed from acid addition of certain organic acids, such as organiccarboxylic acids, and inorganic acids, such as alkylsulfonic acids orhydrogen halide acids, to acidic or basic centers on inventioncompounds, such as basic centers on the invention pyrimidine baseanalogs. Metal salts include ones containing Na⁺, Li⁺, K⁺, Ca⁺⁺ or Mg⁺⁺.Other metal salts may contain aluminum, barium, strontium, cadmium,bismuth, arsenic or zinc ion.

Salt(s) of invention compounds may comprise a combination of appropriatecations such as alkali and alkaline earth metal ions or ammonium andquaternary ammonium ions with the acid anion moiety of the phosphoricacid or phosphonic acid group, which may be present in inventionpolymers or monomers.

Salts are produced by standard methods, including dissolving free basein an aqueous, aqueous-alcohol or aqueous-organic solution containingthe selected acid, optionally followed by evaporating the solution. Thefree base is reacted in an organic solution containing the acid, inwhich case the salt usually separates directly or one can concentratethe solution.

Suitable amine salts include amines having sufficient basicity to form astable salt, preferably amines of low toxicity including trialkyl amines(tripropylamine, triethylamine, trimethylamine), procaine,dibenzylamine, N-benzyl-betaphenethylamine, ephenamine,N,N'-dibenzylethylenediamine, N-ethylpiperidine, benzylamine anddicyclohexylamine.

Salts include organic sulfonic acid or organic carboxylic acid salts,made for example by addition of the acids to basic centers, typicallyamines. Exemplary sulfonic acids include C₆₋₁₆ aryl sulfonic acids,C₆₋₁₆ heteroaryl sulfonic acids and C₁₋₁₆ alkyl sulfonic acids such asphenyl sulfonic acid, a-naphthalene sulfonic acid, β-naphthalenesulfonic acid, (S)-camphorsulfonic acid, methyl (CH₃ SO₃ H), ethyl (C₂H₅ SO₃ H), n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl,pentyl and hexyl sulfonic acids. Exemplary organic carboxylic acidsinclude C₁₋₁₆ alkyl, C₆₋₁₆ aryl carboxylic acids and C₄₋₁₆ heteroarylcarboxylic acids such as acetic, glycolic, lactic, pyruvic, malonic,glutaric, tartaric, citric, fumaric, succinic, malic, maleic, oxalic,hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic,salicylic, nicotinic and 2-phenoxybenzoic.

Invention salts include those made from inorganic acids, e.g., HF, HCl,HBr, HI, H₂ SO₄, H₃ PO₄, Na₂ CO₃, K₂ CO₃, CaCO₃, MgCO₃ and NaClO₃.Suitable anions, which are optionally present with a cation such a Ca⁺⁺,Mg⁺⁺, Li⁺, Na⁺ or K⁺, include arsenate, arsenite formate, sorbate,chlorate, perchlorate, periodate, dichromate, glycodeoxycholate,cholate, deoxycholate, desoxycholate, taurocholate, taurodeoxycholate,taurolithocholate, tetraborate, nitrate, nitrite, sulfite, sulfamate,hyposulfite, bisulfite, metabisulfite, thiosulfate, thiocyanate,silicate, metasilicate, CN⁻, gluconate, gulcuronate, hippurate, picrate,hydrosulfite, hexafluorophosphate, hypochlorite, hypochlorate, borate,metaborate, tungstate and urate.

Salts also include the invention compound salts with one or more aminoacids. Many amino acids are suitable, especially the naturally-occurringamino acids found as protein components, although the amino acidtypically is one bearing a side chain with a basic or acidic group,e.g., lysine, arginine, histidine or glutamic acid, or a neutral groupsuch as glycine, serine, threonine, alanine, isoleucine, or leucine.

The invention compositions include compounds in their un-ionized, aswell as zwitterionic form, and combinations with stoiochimetric amountsof water as in hydrates.

Stereoisomers

The compounds of the invention include enriched or resolved opticalisomers at any or all asymmetric atoms as are apparent from thedepictions. Both racemic and diasteromeric mixtures, as well as theindividual optical isomers isolated or synthesized so as to besubstantially free of their enantiomeric or diastereomeric partners, areall within the scope of the invention. Chiral centers may be found ininvention compounds at, for example, R¹, R² and R²¹.

One or more of the following enumerated methods are used to prepare theenantiomerically enriched or pure isomers herein. The methods are listedin approximately their order of preference, i.e., one ordinarily shouldemploy stereospecific synthesis from chriral precursors beforechromatographic resolution before spontaneous crystallization.

Stereospecific synthesis is described in the examples. Methods of thistype conveniently are used when the appropriate chiral starting materialis available and reaction steps are chosen do not result in undesiredracemization at chiral sites. One advantage of stereospecific synthesisis that it does not produce undesired enantiomers that must be removedfrom the final product, thereby lowering overall synthetic yield. Ingeneral, those skilled in the art would understand what startingmaterials and reaction conditions should be used to obtain the desiredenantiomerically enriched or pure isomers by stereospecific synthesis.If an unexpected racemization occurs in a method thought to bestereospecific then one needs only to use one of the followingseparation methods to obtain the desired product.

If a suitable stereospecific synthesis cannot be empirically designed ordetermined with routine experimentation then those skilled in the artwould turn to other methods. One method of general utility ischromatographic resolution of enantiomers on chiral chromatographyresins. These resins are packed in columns, commonly called Pirklecolumns, and are commercially available. The columns contain a chiralstationary phase. The racemate is placed in solution and loaded onto thecolumn, and thereafter separated by HPLC. See for example, ProceedingsChromatographic Society--International Symposium on Chiral Separations,Sep. 3-4, 1987. Examples of chiral columns that could be used to screenfor the optimal separation technique would include Diacel Chriacel OD,Regis Pirkle Covalent D-phenylglycine, Regis Pirkle Type 1A, AstecCyclobond II, Astec Cyclobond III, Serva Chiral D-DL=Daltosil 100,Bakerbond DNBLeu, Sumipax OA-1000, Merck Cellulose Triacetate column,Astec Cyclobond I-Beta, or Regis Pirkle Covalent D-Naphthylalanine. Notall of these columns are likely to be effective with every racemicmixture. However, those skilled in the art understand that a certainamount of routine screening may be required to identify the mosteffective stationary phase. When using such columns it is desirable toemploy embodiments of the compounds of this invention in which thecharges are not neutralized, e.g., where acidic functionalities such ascarboxyl are not esterified or amidated.

Another method entails converting the enantiomers in the mixture todiasteriomers with chiral auxiliaries and then separating the conjugatesby ordinary column chromatography. This is a very suitable method,particularly when the embodiment contains free carboxyl, amino orhydroxyl that will form a salt or covalent bond to a chiral auxiliary.Chirally pure amino acids, organic acids or organosulfonic acids are allworthwhile exploring as chiral auxiliaries, all of which are well knownin the art. Salts with such auxiliaries can be formed, or they can becovalently (but reversibly) bonded to the functional group. For example,pure D or L amino acids can be used to amidate the carboxyl group ofembodiments of this invention and then separated by chromatography.Enzymatic resolution is another method of potential value. In suchmethods one prepares covalent derivatives of the enantiomers in theracemic mixture, generally lower alkyl esters (for example of carboxyl),and then exposes the derivative to enzymatic cleavage, generallyhydrolysis. For this method to be successful an enzyme must be chosenthat is capable of stereospecific cleavage, so it is frequentlynecessary to routinely screen several enzymes. If esters are to becleaved, then one selects a group of esterases, phosphatases, andlipases and determines their activity on the derivative. Typicalesterases are from liver, pancreas or other animal organs, and includeporcine liver esterase.

If the enatiomeric mixture separates from solution or a melt as aconglomerate, i.e., a mixture of enantiomerically-pure crystals, thenthe crystals can be mechanically separated, thereby producing theenantiomerically enriched preparation. This method, however, is notpractical for large scale preparations and is of no value for trueracemic compounds.

Asymmetric synthesis is another technique for achieving enantiomericenrichment. For example, a chiral protecting group is reacted with thegroup to be protected and the reaction mixture allowed to equilibrate.If the reaction is enantiomerically specific then the product will beenriched in that enantiomer.

Further guidance in the separation of enantiomeric mixtures can befound, by way of example and not limitation, in "Enantiomers, Racemates,and resolutions", Jean Jacques, Andre Collet, and Samuel H. Wilen(Krieger Publishing Company, Malabar, Fla., 1991, ISBN 0-89464-618-4):Part 2, Resolution of Enantiomer Mixture, pages 217-435; moreparticularly, section 4, Resolution by Direct Crystallization, pages217-251, section 5, Formation and Separation of Diastereomers, pages251-369, section 6, Crystallization-Induced Asymmetric Transformations,pages 369-378; and section 7, Experimental Aspects and Art ofResolutions, pages 378-435; still more particularly, section 5.1.4,Resolution of Alcohols, Transformation of Alcohols into Salt-FormingDerivatives, pages 263-266, section 5.2.3, Covalent Derivatives ofAlcohols, Thiols, and Phenols, pages 332-335, section 5.1.1, Resolutionof Acids, pages 257-259, section 5.1.2, Resolution of Bases, pages259-260, section 5.1.3, Resolution of Amino Acids, page 261-263, section5.2.1, Covalent Derivatives of Acids, page 329, section 5.2.2, Covalentderivatives of Amines, pages 330-331, section 5.2.4, CovalentDerivatives of Aldehydes, Ketones, and Sulfoxides, pages 335-339, andsection 5.2.7, Chromatographic Behavior of Covalent Diastereomers, pages348-354.

Compounds of Structure (1)--Polycyclic Substructure

R² is a key functionality. It is substituted on the polycycle depictedin structure (1), less R². The combination of the polycycle and R² istermed the polycyclic substructure. R² consists of two principalstructural features denominated --A(Z)_(X1). Group A is a spacer that isused to position the Z group(s) and attach it to the remainder of thepolycycle. The Z group(s) serve as a site for attachment of a detectablelabel or to enhance the hydrogen bonding of the polycycle to thecomplementary guanine base. In some embodiments, Z is capable ofperforming both functions. For the most part, Z groups capable ofhydrogen bonding are useful as sites for covalent bonding to detectablelabels, but not all Z groups that are useful as label-bonding sites arecapable of hydrogen bonding to guanine. Z contains at least one atomother than carbon, typically O, N or S. In any case, the R² groupspossess at least one of these practical utilities. It would be routineto make and test them to determine the best use of any one embodiment.

Z groups capable of base-pairing are believed to hydrogen bond with N⁷of guanine in a complementary nucleic acid sequence when incorporatedinto a polycyclic substructure-substituted oligonucleotide. Theresulting duplex has greater stability than one containing a native GCpair because the R² group provides an additional point for hydrogenbonding to the complementary guanine base. Thus, these embodiments serveas cytosine surrogates for supplemented Watson-Crick base-pairing. Ingeneral, a base-pairing substituent Z is defined functionally as anygroup that, when taken together with the remainder of R², is capable ofincreasing the temperature of melting of any of the oligonucleotides 3-9in Table 1 by at least about 2 degrees Centigrade when substituted asshown in Table 1.

If R² does not contain a substituent that is capable of contributing tobase pairing or hydrogen bonding then R² is useful at least as a pointof attachment for a detectable label. Such Z groups need only bereactive with a bifunctional cross-linking agent or with the labeldirectly. In some embodiments, the polycyclic substructure is itselffluorescent, and in these cases it is not necessary to link the Z groupto a detectable label. In these embodiments the polycyclic substructureis detectable by fluorescent emissions, or by adsorption and energytransfer to an emitting (second) label present on a binding partner inthe same fashion as is used in EMIT technologies well-known in thediagnostics field.

Spacer A is substituted with from 1 to 3 Z groups. When Z is abase-pairing hydrogen bonding group then "X1" is preferably 1 or 2,ordinarily 1. Similarly, for reasons of steric access it is preferredthat only 1 or 2 Z groups are present on spacer A when R² is intended tofunction as a label bonding site.

In some embodiments the spacer group A and the Z substituent(s) willinteract functionally, i.e., changes in group A may have an impact onthe physical or chemical properties of Z, and vice-versa. For example,it will be understood by those skilled in the art that changes can beintroduced in spacer A that would reduce or increase the ability of Z tohydrogen bond or to react with a label or cross-linking agent. A readilyapparent instance of this would be substitution of A with electrondonors or acceptors proximal to a Z group, which may affect hydrogenbonding between Z and guanine. However, it is conceptually useful toconsider these domains to be functionally and structurally discretetaking into account interdomain interactions that would be apparent tothe ordinary artisan.

Spacer A typically contains a backbone chain of 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or 16 carbon atoms, any 1, 2 or 3 of which areoptionally replaced with N, O or S atoms, usually 1 N, O or S atom. Thebackbone chain refers to the atoms that connect the Z group(s) to thering carbon atom at the R² binding site on the polycycle. The number ofspacer backbone atoms does not include terminal Z group atoms. R² doesnot include protected amine as described in U.S. Pat. No. 5,502,177.

The spacer A backbone is linear or one or more backbone atoms aresubstituted, which results in branching. Ordinarily, when 1 Z group ispresent then A will contain a linear backbone of 2 to 8, usually 2 to 4atoms. The backbone generally is carbon only, bonded by saturated orunsaturated bonds. If unsaturated bonds are present, the backbonegenerally will contain 1 to 2 double or triple bonds. Preferably, thebackbone is saturated. If a heteroatom is present in the backbone ittypically will be O or S. Preferably the heteroatom is O, and preferablyonly 1 O is present in the backbone chain. Heteroatoms are used toreplace any of the backbone carbon atoms, but preferably are used toreplace the carbon atom alpha (adjacent) to the polycyclic ring. Usuallythe atom in the spacer chain that is bonded to the polycyclicsubstructure is unsubstituted, e.g., --O--, --S--, --NH-- or --CH₂ --,and, in general, the next 1, 2 or 3 atoms in the spacer areunsubstituted carbon.

The spacer A backbone is optionally substituted independently with 1, 2or 3 of the following: C₁ -C₈ alkyl, --OR⁵, ═O, --NO₂, --N₃, --COOR⁵,--N(R⁵)₂, or --CN groups, C₁ -C₈ alkyl substituted with --OH, ═O, --NO₂,--N₃, --COOR⁵, --N(R⁵)₂, or --CN groups, or any of the foregoing inwhich --CH₂ -- is replaced with --O--, --NH-- or --N(C₁ -C₈ alkyl),wherein R⁵ is H or a protecting group. Certain of these groups mayfunction as Z sites for linking to detectable labels, but need not beused for that purpose unless desired. In some embodiments thesesubstituents are useful in increasing the lipophilicity of the compoundsof this invention.

Group Z detectable labels include all of the conventional assayablesubstances used heretofore in labeling oligonucleotides or proteins.Examples are well known and include fluorescent moieties such asfluorescein, chemiluminescent substances, radioisotopes, chromogens, orenzymes such as horseradish peroxidase. For the purposes herein, theresidue of any bifunctional or multifunctional agent used to crosslinkthe Z group(s) to the A backbone is defined to be part of the Z group,and the residue of the detectable label is considered also to representpart of Z.

Group Z also encompasses substituents that are not detectable byconventional diagnostic means used in clinical chemistry settings (e.g.,UV or visible light absorption or emission, scintillation or gammacounting, or the like) but which are nonetheless capable of reactingwith a crosslinking agent or a detectable label to form a covalent bond.In this regard, the Z groups function as intermediates in the synthesisof the labelled reagent. Typical Z groups useful for this purposeinclude --NH₂, --CHO, --SH, --CO₂ Y or OY, where Y is H,2-hydroxypyridine, N-hydroxysuccinimide, p-nitrophenyl, acylimidazole,maleimide, trifluoroacetate, an imido, a sulfonate, an imine1,2-cyclohexanedione, glyoxal or an alpha-halo ketone. Suitable spacers,reactive groups and detectable labels have been described, e.g., U.S.Pat. Nos. 5,668,266, 5,659,022, 5,646,261, 5,629,153, 5,525,465 and5,260,433, WO 88/10264, WO 97/31008, EP 063 879 B1, Urdea "NAR"16:4937-4956 (1988), Prober "Science" 238:336-341 (1987).

Z also is a hydrogen bond donor moiety or a moiety, when taken togetherwith the influence of spacer A, has a net positive charge of at leastabout +0.5 at pH 6-8 in aqueous solutions. Such Z groups are designatedR^(2D). In these embodiments, R^(2D) is covalently linked to a shortspacer A having a backbone (otherwise described above) of 2, 3, 4, 5 or6 atoms, designated R^(2C).

The R^(2C) short spacer chain backbone atoms are C atoms and optionallyone or two atoms independently selected from the group consisting of O,N or S atoms. R^(2C) short spacer chain backbones include unbranched andbranched alkyl that optionally contain one or two independently selectedO, N or S atoms. Usually R^(2C) is unbranched, i.e. the backbone has nohydrocarbon substituents. Any branching, if present, will usuallyconsist of a C₁ -C₃ alkyl group, usually a methyl or ethyl group, or C₁-C₃ alkyl substituted with --OH, ═O, --O(C₁ -C₃ alkyl), --CN, N₃ or 1,2, 3 or 4 halogen atoms.

Exemplary --R^(2C) --R^(2D) and related structures are

(a) --R⁶ --(CH₂)_(t) --NR⁵ C(NR⁵)(NR³)₂, including --O--(CH₂)_(t) --NR⁵C(NR⁵)(NR³)₂, --NH--(CH₂)_(t) --NR⁵ C(NR⁵)(NR³)₂ and --(CH₂)₂₋₅ NR⁵C(NR⁵)(NR³)₂,

(b) --R⁶ --CH₂ --CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_(v) --N(R³)₂, --R⁶--(CH₂)_(t) N(R³)₂, --(CH₂)_(t) N(R³)₂, --CH₂ --O--(CH₂)_(t) --N(R³)₂,##STR7## where R⁶ is usually --O--, ##STR8## where R⁶ is usually --O--and R⁸ is usually --CH₂ -- or --CH₂ CH₂ -- in structures (42)-(46) andadjacent R⁶ and R⁸ are not --O--O--, --O--S-- or --S--S--;

R³ is independently --H, --CH₃, --CH₂ CH₃, --(CH₂)_(w) --N(R³³)₂ or aprotecting group, usually --H or --CH₃,

or, both R³ together are joined to form a protecting group,

or, when R² is --R⁶ (CH₂)_(t) N(R³)₂, one R³ is H, CH₃, CH₂ CH₃, aprotecting group or --(CH₂)_(w) --N(R³³)₂ and the other R³ is --H,--CH₃, --CH₂ CH₃, --(CH₂)_(w) --N(R³³)₂, --CH(N[R³³ ]₂)--N(R³³)₂,##STR9## usually when one R³ is --H, --CH₃, --CH₂ CH₃, or --(CH₂)_(v)--N(R³³)₂, the other R³ is --H or a protecting group;

R⁵ is independently --H or a protecting group;

R⁶ is independently --S--, --NR⁵ --, --O-- or --CH₂ --;

R⁷ is independently linear alkyl having 1, 2, 3 or 4 carbon atoms,linear alkyl having 2, 3 or 4 carbon atoms and containing one --CH═CH--,--C.tbd.C-- or --CH₂ --O--CH₂ -- moiety, or R⁷ is cyclic alkyl having 3,4 or 5 carbon atoms, wherein one of the linear alkyl carbon atoms isoptionally substituted with a single --CH₃, --CN, ═O, --OH or protectedhydroxyl, provided that the carbon atoms in any --CH═CH-- or --CH₂--O--CH₂ -- moiety are not substituted with ═O, --OH or protectedhydroxyl, and usually R⁷ is --CH₂ --, --CH₂ --CH₂ --, --CH₂ --CH₂ --CH₂-- or --CH₂ --CH₂ --CH₂ --CH₂ --;

R⁸ is linear alkyl having 1 or 2 carbon atoms wherein one of the linearalkylene carbon atoms is optionally substituted with a single --CH₃,--CN, ═O, --OH or protected hydroxyl, or R⁸ is absent and R⁶ is linkeddirectly to the ring in R² structures (42)-(46), usually R⁸ is --CH₂ --or --CH₂ --CH₂ --;

R²⁸ is independently --CH₂ --, --CH(CH₃)--, --CH(OCH₃)--, --CH(OR⁵)-- or--O--, but both are not --O--;

R²⁹ is independently --N--, --N(CH₃)--, --CH--, --C(CH₃)--, but both arenot --N(CH₃)--;

R³⁰ is --H or --N(R³)₂, usually --H or --NH₂ ;

R³¹ is the side chain of an amino acid, usually the side chain of anaturally occurring amino acid, e.g. glycine, alanine, valine,isovaline, leucine, threonine, serine, lysine or arginine;

R³³ is independently --H, --CH₃, --CH₂ CH₃ or a protecting group;

R³⁵ is H, C₁ -C₄ alkyl (including --CH₃, --CH₂ CH₃) or a protectinggroup, usually --H or a protecting group;

R³⁶ is --H, --CH₃, --CH₂ CH₃, a protecting group, a monosaccharide,where the monosaccharide is usually linked at the monosaccharide's 1'position and where any monosaccharide hydroxyl groups are optionallyprotected, typically an R³⁶ monosaccharide is 2'-deoxyribose, a2'-deoxy-2'-R²¹ -substituted ribose or arabinose such as2'-deoxy-2'-fluororibose or 2'-deoxy-2'-fluoroarabinose, or themonosaccharide is ribose or arabinose;

t is 1, 2, 3 or 4, but when R⁶ is --O--, --S-- or --NR⁵ --, t is 2,3 or4;

v is independently 0, 1 or 2; and

w is 1 or 2.

Invention embodiments include R² moieties having the structure --R⁵⁹--NH₂ where R⁵⁹ has the structure --R⁶ --R⁶⁰ --, including --R⁶--(CH₂)_(t) --N(R₃)₂, where R⁶ is usually --O--, --S--, --NH-- or --CH₂--, R⁶⁰ is --CHR⁵¹ --(CHR⁵¹)_(Z3) --(R⁶¹)_(Z1) --(CHR⁵¹)_(Z2) --CHR⁵¹ --where R⁶¹ is --O--, --S--, C(O), --CHR⁵ or --NR⁵ -- and usually 0, 1 or2 R⁵¹ are methyl or ethyl; R⁵¹ independently is --H, methyl or ethyl; Z3is 1, 2 or 3, usually 1; Z1 is 0 or 1, usually 0; and Z2 is 1, 2 or 3,usually 1. In these embodiments, any functional groups, e.g., --OH,--NH₂, --COOH, or --SH, that are optionally present at R²¹ are usuallyprotected. These embodiments are useful as intermediates useful to makemonomers for oligonucleotide synthesis.

Other invention embodiments include R² moieties having the structure##STR10## where R⁶¹ is --H, alkyl having 1, 2, 3 or 4 carbon atoms oroptionally protected substituted alkyl having 1, 2, 3, 4, 5 or 6 carbonatoms including --CH₃ and --CH₂ CH₃, and R⁶² is --H, --NH₂ or --NH(CH₃).Other embodiments are --R⁶ --(CH₂)₂₋₈ --NH₂, --R⁶ --(CH₂)₂₋₈ --OR⁵ or--R⁶ --(CH₂)₂₋₈ --CO₂ R^(5A).

Compounds of Structure (1)--Substituent R¹ --Linker

One uses R¹ linker groups to covalently bond the invention base to theselected binding partner, although it will be understood that this neednot be the sole use for the linker functionality. Thus, a group presentin R¹ linkers principally serves as the site for covalently bonding theinvention base to a binding partner, typically by incorporating theinvention base via the linker residue into a polymeric binding partnerby grafting or copolymerization.

R¹ linkers also optionally are substituted with groups that ordinarilywill not participate in binding to the binding partner, e.g., halo,azido and protected hydroxyl. Generally, such linker groups will containfrom 2 to about 50 atoms. If it contains a cycle the cyclicfunctionality typically will be an oxygen, sulfur orphosphorus-containing saturated or unsaturated heterocycle having atotal of about from 5 to 7 ring atoms and 1 to 3 heteroatoms. For themost part, the cycle will be a monosaccharide, typically (i) a hexose,(ii) a hexose such as glucose substituted with phosphate, protectedphosphate, hydrogen phosphonate, a phosphoramidate, hydroxyl orprotected hydroxyl, (iii) a furanose or (iv) a furanose substituted withphosphate, protected phosphate, hydrogen phosphonate, a phosphoramidate,hydroxyl or protected hydroxyl. Typical furanose sugars include ribose,2'-deoxyribose, 2'-deoxy-2'-R²¹ -substituted ribose and their 2' araisomers. Ordinarily, R¹ is an abasic nucleotide residue or such aresidue derivatized so as to be capable of incorporation into anoligonucleotide.

Thus, the R₁ linker frequently comprises an activated group or othergroup which can react with a polymer or other binding partner to belabeled with the polycyclic substructure. For example, groups describedbelow that are compatible with commonly available oligonucleotidesynthetic chemistries are useful. Other examples of reactant groups forcovalent labeling are well-known from the diagnostic fields and haveheretofore been used commonly to label proteins and oligonucleotideprobes, as is more fully discussed below.

In one embodiment, R¹ is a bifunctional or multifunctional organiclinker group such as alkyl, alkene, alkyne, alkoxyalkyl, alkylthioalkyl,alkoxy, saturated or unsaturated heterocycle that is substituted with atleast one group capable of being crosslinked with or incorporated into apolymer, e.g., such groups as hydroxy, amino, carboxyl, vinyl, phosphateor phosphonate. U.S. Pat. No. 5,502,177 describes suitable linkergroups. An example of such an R¹ linker suitable for oligonucleotidesynthesis is protected monosaccharides, such as ribofuranose anddeoxyribofuranose sugars of structure (5) ##STR11## where an inventionbase is linked to the open valence at the 1' position, D is hydroxyl,protected hydroxyl or is an oligonucleotide coupling group and D¹ isindependently R²¹ or an oligonucleotide coupling group, but both D¹ arenot coupling groups.

In embodiments of the invention where the compound of structure (1) isto be used as a monomer in the preparation of oligonucleotides, R¹ istypically structure (5) where one D¹ is an oligonucleotide couplinggroup and D is --OH or protected hydroxyl.

"Coupling group" as used herein means any group suitable for generatinga phosphodiester linkage or phosphodiester substitute linkage betweennucleotide bases or their analogs. These coupling groups areconventional and well-known for the preparation of oligonucleotides, andare prepared and used in the same fashion here. They are usuallyconfigured as the b anomers as denoted in structure (5) or optionally asthe alpha anomers. In general, each compound comprising structure (5)will contain two coupling groups: D or D¹, but with only one D¹ being acoupling group. The coupling groups are used as intermediates in thepreparation of 3',5' 5',3', 5',2' and 2',5' internucleotide linkages inaccord with known methods.

Suitable coupling groups for phosphodiester linkages or phosphodiestersubstitute linkages containing phosphorus include OH, H-phosphonate;(for amidite chemistries) alkylphosphonamidites or phosphoramidites suchas β-cyanoethylphosphoramidite,N,N-diisopropylamino-β-cyanoethoxyphosphine,N,N-diisopropylamino-methoxyphosphine,N,N-diethylamino-methoxyphosphine,N,N-diethylamino-β-cyanoethoxyphosphine,N-morpholino-β-cyanoethoxyphosphine, N-morpholino methoxyphosphine,bismorpholino-phosphine,N,N-dimethylamino-β-cyanoethylmercapto-phosphine,N,N-dimethylamino-2,4-dichlorobenzylmercaptophosphine, andbis(N,N-diisopropylamino)-phosphine; and (for triester chemistries) 2-,or 4-chlorophenyl phosphate, 2,4-dichlorophenyl phosphate, or2,4-dibromophenyl phosphate. See for example U.S. Pat. Nos. 4,725,677;4,973,679; 4,997,927; 4,415,732; 4,458,066; 5,047,524; 4,959,463;5,624,621; and International Publication Nos. WO 97/14706 and WO92/07864.

For structure (2) embodiments, if D¹ is a coupling group then Dtypically will be hydroxyl protected with a group suitable for ensuringthat the monomer is added to the oligonucleotide rather than dimerizing.Such groups are well known and include DMT, MMT, FMOC(9-fluorenylmethoxycarbonyl), PAC (phenoxyacetyl), a trialkyl (C₁ -C₆alkyl, each alkyl group is independently chosen) silyl ether or an alkyl(C₁ -C₆ alkyl) diaryl (e.g., phenyl) silyl ether such as TBDMS(t-butyldiphenylsilyl) and TMS (trimethylsilyl). The opposite will applywhen one desires to synthesize an oligonucleotide in the oppositedirection (5'→3'). Ordinarily in structure (5) compounds, D is DMT, D¹is located on the 3' carbon, R²¹ is H and the D¹ and R²¹ groups are inthe alpha anomer conformation.

As noted above, R¹ includes an optionally protected monosaccharides ofstructure (4) and (4A). Usually the monosaccharides in structure (4) and(4A) compounds are 2'-deoxyribose, 2'-deoxy-2'-R²¹ -substituted ribose,2'-deoxy-2'-R²¹ -substituted arabinose, ribose or arabinose, any ofwhich are optionally protected at sugar all or some hydroxyls or atoptionally present R²¹ functional groups such as --OH --SH or --NH₂groups.

Invention embodiments include compositions of the formula ##STR12##where B is a structure (3), (4) or (4A) base, R⁵⁶ is a diene or adieneophile, both as defined in WO 97/14706, R⁵⁷ is --OR⁵, a couplinggroup including --OH, H-phosphonate, a phosphoramidite or an optionallyprotected oligonucleotide having a 3'-terminal group selected from acoupling group and --OR⁵ and any reactive moiety in R²¹ is optionallyprotected. R⁵⁶ dienes are independently chosen and include 2,4-hexadieneand 3,5-hexadiene. One or both R⁵⁶ are linked to a solid support such asa crosslinked organic polymer, polystyrene, Tentagel™, polyethyleneglycol or an inorganic oxide such as silica gel, alumina, controlledpore glass or a zeolite. These compositions are useful for makingoligonucleotides containing one or more invention bases.

Invention embodiments include compositions and their isomers of theformula ##STR13## where R⁵⁷ is independently --H, a protecting group orboth R⁵⁷ together are a dihydroxy protecting group, R⁵⁸ is --H or alkylcontaining 1, 2, 3 or 4 carbon atoms and B is a structure (3), (4) or(4A) base. Suitable R⁵ at the nitrogen atom include --H, FMOC and tBOC(t-butyloxycarbonyl) and suitable R⁵ at the carboxyl group include --H,t-butyl and benzyl, see, e.g., WO 97/14709. These compositions areuseful for making oligonucleotides containing one or more inventionbases.

Protecting groups suitable for use with amine groups that may be presentat R² include FMOC and trichloroacetamide. Monomers and polymers maycontain such protecting groups at R².

Substituent R¹ --Binding Partner

R¹, when functioning as a binding partner, is a substance thatnon-covalently binds to a target compound. Generally, the targetcompound is an analyte whose presence is desired to be detected. Bindingpartners are well-known from the immunoassay art and includehapten-antibody pairs such as those used in drug immunoassays using EMITor ELISA technologies. Binding partners are employed analytically inenzymology, where the substrate or the enzyme is labeled. Bindingpartners also are known from the oligonucleotide hybridization art,including oligonucleotide-nucleic acid binding partners (as indiagnostic probes or therapeutic antisense oligonucleotides) oroligonucleotide-protein binding partners (aptamers). In accordance withthis invention, an invention base is substituted at R¹ by any bindingpartner. While the binding partner may be a small molecule such as adrug, hapten, substrate or the like, ordinarily it is a polymer.

Compounds of structure (1) wherein R¹ is a polymer are an importantfeature of this invention. For the most part, when R¹ is a polymer an R¹linker group has been subsumed into the polymer structure, either as amonomer unit or by grafting onto pre-existing polymer. Therefore, whenR¹ is a polymer, the polymer may comprise the residue of a linking groupderived from a monomer or where the linking group differs from thepolymer's monomeric subunits. The invention base must be covalentlylinked to the polymer.

The nature of the polymer is not critical. Typical R¹ polymers include abiopolymer such as an oligonucleotide, a protein (including antibodies,enzymes, cell membrane proteins, glycoproteins, glycolipids,lipoproteins and nucleoproteins), a peptide, a nucleic acid, or a glycanor other polysaccharide or carbohydrate. In certain embodiments thepolymer is an oligonucleotide in which either or both of the sugar orphosphodiester monomer subunits are substituted by groups that continueto permit base pairing by the invention base analogs but which haveother desirable characteristics that are not shared with nativesubstituents, e.g., those which mask the negative charges of thephosphodiester linkages or replace the phosphodiester linkage withanother group.

The site at which one links the invention base analogs to a polymer istypically not critical. In general, any reactive group on the polymer issatisfactory when one wants to graft the polycycle-R² substructure ontoa pre-existing polymer. Obviously, the site of the substitution shouldnot be in a location in which the polycycle-R² substructure willinterfere with the intended function for the polymer, e.g. enzyme activesite, antibody CDR, and the like as will be understood by the artisan.An amino acid side chain such as that of lysine, glutamic acid, serine,asparagine and the like will be satisfactory for grafting to protein R¹,as will alpha amino groups, provided that the amino acids in question donot participate in the binding partner or ligand/substrate interactioninvolved in the assay in which the labeled protein is to be used. Oneapplies the same reasoning to select a binding site or sites on otheranalytes such as sugars, glycans, lipids, and the like. For example, the1' position of ribose or deoxyribose is satisfactory as the site ofsubstitution of an oligonucleotide by the invention base analogs.Suitable sites will be known to the artisan, particularly in thoseinstances where the one intends to substitute an invention base analogfor purine or pyrimidine bases, usually for cytosine, or for fluorescentlabels.

The degree of polymer substitution by the invention base analogs is notcritical. One skilled in the art will choose the reaction conditionssuch that the resulting labeled polymer will be substituted withsufficient molar proportion of base analog to facilitate its use in thedesired analytical, therapeutic or preparative procedure. This isaccomplished by preparing the labeled polymers under a variety ofconventional conditions, e.g., the time, temperature or duration of thelabeling reaction, to yield a matrix of multiply-labeled polymers. Thesethen are screened for suitability in the intended application. Molarratios of about from 1:1 to 10:1 invention base to polymer generally aresuitable. Where the labeled polymer is prepared by monomerincorporation, the resulting polymer may contain about from 1% to 100%invention base analog substitution. In this embodiment each inventionbase is considered a monomer unit (even though the polymer may have beenassembled from intermediate synthons containing 2 or more inventionbases per synthon).

Oligonucleotides are polymers containing at least 2 covalently linkednucleotides or nucleotide analogs (collectively monomers), at least oneof which comprises an invention base. In oligonucleotide inventionembodiments at least one invention base is covalently linked to anucleotide sugar, and typical invention oligonucleotides will containabout 2-75% of the bases as invention base analogs, usually about 5-25%.Small oligonucleotides, e.g., 2-6-mers, that serve as syntheticintermediates for larger oligonucleofides will optionally contain thehigher proportions of invention base analogs, e.g., about 50-75%. Largeroligonucleotides, e.g., about 7-21-mers, will generally contain 1, 2, 3,or 4 invention bases, occasionally 5 and usually not more than about 5invention bases, unless the oligonucleotide is relatively long, e.g.,about 22-50-mer.

Invention embodiments include polymers and oligonucleotides where theinvention bases are located on 2, 3 or more adjacent monomers ornucleotide residues, or the invention bases may be located on monomersor nucleotide residues that are separated from each other by about 1, 2,3, 4, 6, 8, 10, 12, 15, 18 or more monomers or nucleotide residues thatdo not contain these bases. When a detectable label is linked to aninvention base at R², the oligonucleotide may contain 1, 2 or 3 of theselabeled monomers, usually 1 or 2. Such labelled monomers are oftenlocated at the 3' or 5' terminus, but they may reside at an internalposition such as one, two or more monomer residues from either terminus.

Invention oligonucleotides, which contain 1, 2, 3 or more inventionbases, will typically have sufficient binding affinity for complementarynucleic acid sequences to allow facile detection of the duplex ortriplex resulting from the base sequence-specific binding interaction.Typically, an invention oligonucleotide will have a Tm of at least about15° C., usually at least about 20° C., when tested under typical invitro binding conditions, such as those described herein and elsewhere,(Jones, "J Org Chem" [hereafter "JOC" ] 58:2983-91 1993, Froehler, "Tet.Lett." 34:1003-06 1993). Complementary nucleic acid means a natural orsynthetic compound that is capable of forming a hydrogen bonded complexin a sequence-specific manner with an invention oligonucleotide such asa structure (2) oligonucleotide. Complementary nucleic acid basesequences contain no mismatches, while "substantially complementary"base sequences contain only a limited number of mismatches, e.g., atmost about 1 mismatch per about 15-20 bases, relative to an inventionoligonucleotide.

One optionally measures the binding of an invention oligonucleotide to acomplementary nucleic acid by detecting or measuring a Tm, by detectingthe presence of a label present on the invention oligonucleotide or onthe complementary nucleic acid (after separating bound inventionoligonucleotide from unbound invention oligonucleotide), by amplifyingnucleic acids containing a region(s) complementary to an inventionoligonucleotide and so forth. Because of this, inventionoligonucleotides optionally include species containing one or moremodifications that decrease binding affinity, while the oligonucleotidestill retains sufficient binding affinity for a given application. Inaddition, embodiments include short oligonucleotides or oligonucleotidedomains, e.g., having about 2, 3, 4, 5 or 6 linked monomers, where thedomain may have low binding affinity, but even in this case are usefulas intermediates to make longer oligonucleotides so as to increaseaffinity sufficiently to confer a Tm of at least about 15° C. Generally,invention oligonucleotide analogs will contain about 40% or less,usually about 25% or less, of monomers that significantly reduce bindingaffinity, i.e., monomers that decrease the Tm more than about 2° C. permonomer, compared to a corresponding unmodified oligonucleotide.

Invention embodiments include protected, partially protected anddeprotected monomers and polymers including oligonucleotides. Partiallyprotected compounds arise during the course of deprotection and they arethus intermediates in the process of preparing deprotected compounds.Typically, one would not recover partially deprotected compounds.Deprotected compounds have been subject to a treatment that removes theprotecting group(s), although the preparation may contain some compoundswith unremoved protecting groups. Typically, any remaining protectinggroups that remain after deprotection are present in small amounts thatmay be removed by suitable purification methods if desired.

Invention embodiments include oligonucleotides of structure (2) whereR³⁷ is oxygen. Invention oligonucleotides, including those where R³⁷ isoxygen, typically contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 linked monomers usually about5-21. Such oligonucleotides optionally contain about 30-100%, typicallyabout 60-100%, of the linkages as phosphodiester or phosphorothioatelinkages, or other linkages of similar binding affinity.

Invention oligonucleotides include support-bound oligonucleotides, whichare typically used in solid phase synthesis and separation applications.Support-bound oligonucleotides are typically protected during synthesis,e.g., bases, sugar hydroxyls, linkages and functional groups optionallypresent are protected as needed, e.g., a sugar hydroxyl group present atR¹, an amine group at R² or a hydroxyl or amine group at R²¹. In theseembodiments, R¹ is covalently linked to a solid support or R¹ is anoligonucleotide linked to a solid support. When one removes inventionoligonucleotides from a support, the protecting groups are generallyremoved at the same time or shortly thereafter.

Invention embodiments include highly lipophilic polymers andoligonucleotides that comprise (i) one or more structure (3) bases,usually about 1, 2, 3 or 4, and (ii) lipophilic modifications such thatthe polymer or oligonucleotide has an octanol:water partitioncoefficient of about -0.5 to about 2.5, typically about 0.0-2.0, usuallyabout 0.2-1.5, and a solubility in water of at least 0.001 μg/mL,usually at least 0.1 μg/mL.

One can use such highly lipophilic polymers and oligonucleotides asreagents to stain, detect or visualize living cells in vitro or in vivo,as described in U.S. Pat. No. 5,633,360 and in Application No. PCT US96/12530. These highly lipophilic polymers and oligonucleotides need notbe binding competent for cell staining, detecting or visualizingapplications and they are optionally labeled using standard labels,e.g., radiolabels (³² p, ³⁵ S, ¹³¹ I, ¹⁴ C, ³ H), fluorescent labelssuch as fluorescein, Texas Red, rhodamine, BODIPY, resorufin orarylsulfonate cyanines and chemiluminescent labels, e.g., acridiniumesters.

Embodiments of such optionally labeled highly lipophilic inventionoligonucleotides include species where (i) at least about 30%, typicallyat least about 40%, usually at least about 60% (often at least about80%), of the internucleotide linkages are non-ionic internucleotidelinkages (typically containing a lipophilic moiety at each non-ioniclinkage), or (ii) at least about 30%, typically at least about 40%usually at least about 60%, of the bases included in saidoligonucleotide contain a lipophilic substitution, (iii) at least about30%, typically at least about 40% usually at least about 60%, of thesugars, usually at the 2' position, included in said oligonucleotidecontain a lipophilic substitution or (iv) the percent non-ionicnucleotide linkage and the percent lipophilic bases and the percentlipophilic sugars sum to at least about 30%, typically at least about40% usually at least about 60% (or at least about 80%).

Usually, the invention base analogs and other noninvention bases thatare present in binding-competent oligonucleotides are linked together byan organic moiety that is sufficiently flexible to permit the inventionbase analog(s) to hybridize to complementary bases. The linkage may be aconventional phosphodiester linkage in which a nucleotide analogcontaining a structure (1) compound, where R¹ is deoxyribosyl, ribosylor an analog thereof, which is incorporated into an oligonucleotide byconventional methods. Alternatively, other groups are used to replacethe phosphodiester linkage or, in some instances, both of thephosphodiester linkage and the sugar group. These replacement groups aretermed "phosphodiester substitute linkages" for the purposes herein.

Phosphodiester substitute linkages are well-known from the priorliterature. They include for example phosphorodithioates (Marshal,"Science" 259:1564, 1993), phosphorothioates and alkylphosphonates (U.S.Pat. No. 5,212,295, Kibler-Herzog, "Nucleic Acids Research" [hereafter"NAR" ] 19:2979, 1991; PCT 92/01020; EP 288,163; FIG. 12-1),phosphoroamidates (Froehler, "NAR" 16:4831, 1988), 3'-NHphosphoramidates (Schultz, "NAR" 24:2966, 1996; Gryaznov, "J Am ChemSoc" [hereafter "JACS"] 116:3143, 1994; Chen, "NAR" 22:2661, 1995;Gryaznov, "Proc Natl Acad Sci" USA 92:5798, 1995), phosphotriesters(Marcus-Sekura, "NAR" 15:5749, 1987), boranophosphates (Sood, "JACS"112:9000, 1991), 3'-O-5'-S- phosphorothioates (Mag, "NAR" 19:1437,1991), 3'-S-5'-O-phosphorothioates (Kyle, Biochemistry 31:3012, 1992),3'-CH₂ -5'-O-phosphonates (Heinemann, "NAR" 19:427, 1991),3'-NH-5'-O-phosphonates (Mag, "Tet. Lett." 33:7323, 1992), sulfonatesand sulfonamides (Reynolds, "JOC" 57:2983, 1992), sulfones (Huie, "JOC"57:4519, 1992), sulfoxides (Huang, "JOC" 56:3869, 1991), sulfides(Schneider, "Tet Lett." 30:335, 1989), sulfamates, ketals andformacetals (Matteucci, "JACS" 113:7767, 1991, PCT 92/03385 and PCT90/06110), 3'-thioformacetals (Jones, "JOC" 58:2983, 1993),5'-S-thioethers (Kawai, "Nucleosides Nucleotides" 10:1485, 1991),carbonates (Gait, "J Chem Soc Perkin Trans 1" 1389, 1979), carbamates(Stirchak "JOC" 52:4202, 1987), hydroxylamines (Vasseur, "JACS"114:4006, 1992), methylamine (methylimines) and methyleneoxy(methylimino) (Debart, "Bioorg Med Chem Lett" 2:1479, 1992) and amino(PCT 91/06855). Also of interest are hydrazino and siloxane (U.S. Pat.No. 5,214,134) linkages, thionotriester linkages (WO 96/29337) andrelated synthesis methods (WO 97/31009).

Phosphodiester substitute linkages per se also are known for thereplacement of the entire phosphoribosyl linkage of conventionaloligonucleotides. These include for example morpholino-carbamates(Stirchak, "NAR" 17:6129, 1989), peptides (Nielsen et al., "Science"254:1497, 1991; U.S. Ser. Nos. 07/892,902 and 07/894, 397), riboacetallinkages (PCT 92/10793) and morpholino-based linkages disclosed in U.S.Pat. Nos. 5,521,063 and 5,185,144.

Additional disclosure of phosphodiester substitute linkages is found inU.S. Pat. No. 5,386,023, U.S. Pat. No. 5,489,677, WO 95/18623, WO94/00467, WO 93/08296, WO 92/20822, WO 92/20823, PCT 91/08213, 90/15065,91/15500, 92/20702, 92/20822, 92/20823, 89/12060 and 91/03680; Mertes,"J Med Chem" 12:154, 1969; Mungall, "JOC" 42:703, 1977; Wang, "Tet Lett"32:7385, 1991; Stirchak, "NAR" 17:6129, 1989; Hewitt, "Nucleosides andNucleotides" 11:1661, 1992; Van Aerschot, "Agnew Chem Int Ed Engl"34:1338, 1995; and U.S. Pat. Nos. 5,034,506 and 5,142,047.

Invention embodiments include oligonucleotides having 1, 2, 3 or moreoptionally protected invention bases, 0 to about 30 other optionallyprotected bases, usually guanosine, adenine, thymine, cytosine or5-methylcytosine, and at least one modified linkage, e.g., 3'--N(R¹¹)--O-- 5', where R¹¹ is hydrogen, C₁₋₆ alkyl, usually --CH₃ or--C₂ H₅ and the terminal atoms are linked to the 3' and 5' carbons ofadjacent ribose or 2'-deoxy-2'-R²¹ substituted ribose sugars, ##STR14##where R³⁸ independently is O, CH₂ or NH; R⁴⁰ independently is hydrogen,C₁₋₈ alkyl (methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,t-butyl, etc.), a protecting group or both R⁴⁰ together with thenitrogen atom to which they are attached form ##STR15## or both R⁴⁰together are a protecting group, or R⁴⁰ is alkyl (C₁ -C₁₂), usuallyunbranched or branched once containing 1, 2, 3, 4, 5, 6, 7 or 8 carbonatoms, including methyl, ethyl, n-propyl and isopropyl) or R⁴⁰ issubstituted alkyl (C₁ -C₁₂, usually unbranched or branched oncecontaining 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, with substituentsincluding one, two or more --O--, --C(O)--, --OC(O)--, --C(O)O--,--OR⁴², --SR⁴³, --C(O)NR³⁹ --, --C(O)N(R⁴¹)₂, --NR⁴¹ --, --N(R⁴¹)₂, halo(e.g., --F, --Cl), --CN, --NO₂ moieties); R⁴¹ independently is hydrogen,a protecting group (or both R⁴¹ together are a protecting group), alkyl(C₁ -C₄ including methyl, ethyl and n-propyl); R⁴² is hydrogen or aprotecting group; R⁴³ is C₁₋₆ alkyl or a protecting group; and R⁴⁵ is--H, a counter ion or a hydrolyzable moiety such as ##STR16## R⁴⁶ isalkyl containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. R⁴⁰ pairsinclude ones where one R⁴⁰ is hydrogen and the other R⁴⁰ is alkylcontaining 1, 2, 3, 4, 5 or 6 carbon atoms or substituted alkylcontaining 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, including methyl,ethyl, methoxyethyl and ethoxyethyl. When R⁴⁰ is substituted alkyl, itwill usually contain 1, 2, 3 or 4 non-carbon atoms, but may containadditional non-carbon atoms, particularly when the non-carbon atoms arehalogens or when a group is present as a protecting group, e.g., R³⁹,R⁴⁰, R⁴¹ or R⁴².

Structure (2) and (52) oligonucleotides include species where one ormore R²¹ is --F, --O(CH₂)₂ NHR⁵, --O(CH₂)₃ NHR⁵, --O(CH₂)₄ NHR⁵,--O(CH₂)₂ OCH₃, --O(CH₂)₃ OCH₃, --O(CH₂)₂ OR⁵, --O(CH₂)₂ F, --O(CH₂)₃OR⁵, or --O(CH₂)₃ F (see, e.g., Griffey "J Med Chem" 39:5100-5109 1996,Schultze "Cell" 24:2966-2973 1996). Such oligonucleotides includeoligonucleotides where 1, 2, 3, 4, 5, 6, 7, 8 or more monomers aresubstituted with R²¹, which will optionally comprise one of thesesubstituents and the remaining R²¹ are all hydrogen. Embodiments alsoinclude optionally protected monomers containing an optionally protectedinvention base for synthesis of phosphoramidate-linked oligonucleotides.Oligonucleotides containing one or more of these linkages are optionallyprepared as highly lipophilic oligonucleotides and they are suitable forcell staining uses, diagnostic uses and for antisense applications thatoptionally rely at least in part on an RNase H mechanism.

Invention embodiments include oligonucleotides or monomers described inU.S. Pat. Nos. 5,670,489, 5,667,976, 5,652,355, 5,652,356 and 5,212,295where one or more optionally protected invention bases is present,usually 1, 2, 3 or 4.

Invention embodiments include oligonucleotides having 1, 2, 3 or moreoptionally protected invention bases, 0 to about 30 other bases(optionally protected) and at least one amide linkage, e.g., a compoundof structure (2B) where n is 0 to about 50, usually about 5-21. Suchamide linkages have been described, e.g., (Haaima "Agnew Chem Int EdEngl" 35:1939-1942 1996; Nielsen "Bioconjugate Chem" 5:3-7 1994). Otheramide linkages that are suitable have been described, e.g., WO 92/20702and WO 93/24507. Embodiments also include optionally protected monomerscontaining an optionally protected invention base for synthesis ofamide-linked oligonucleotides. In general, structure (2B)oligonucleotides will contain only amide linkages, but they may alsocomprise a domain of monomers linked by non-amide linkages. Suitable D²and D³ have been described, e.g., WO 86/05518, WO 92/20702, WO 93/24507.D² optionally comprises a peptide coupling group, a protecting group, ora solid support. D³ optionally comprises --H, a peptide coupling group,a protecting group, or a solid support, but D² and D³ are not both apeptide coupling group or a solid support. WO 92/20702 describedactivated derivatives of --CO₂ H and --SO₃ H.

The phosphodiester or phosphodiester substitute linkages herein are usedto bond the 2' or 3' carbon atoms of ribose or ribose analogs to the 5'carbon atoms of the adjacent ribose or ribose analog. Ordinarily, thelinkages in oligonucleotides are used to bond the 3' atom of the 5'terminal oligonucleotide to the 5' carbon atom of the next 3'-adjacentnucleotide or its analog. In general, linkages that contain a phosphorusatom will be 3',5' linkages and not 2',5' linkages because such linkagesusually confer reduced binding affinity on the oligonucleotide in whichthey are present.

Table 1 of U.S. Pat. No. 5,502,177 describes examples of suitablephosphodiester substitute linkages for use with the invention baseanalogs. The starting materials in Table 1, or those used to prepare thestarting materials of Table 1, generally possess structure (1) in whichR¹ is ribose, 2'-deoxyribose, a ribose analog or a 2'-deoxyribose analogcomprising a 5' hydroxyl group and a 3' or 2' hydroxyl group, preparedas described herein or in the citations, with an invention baseanalog(s) being substituted for the bases used in the citations. Thereactions are repeated or ganged with phosphodiester or other linkagesin order to produce trimers, tetramers, pentamers or largeroligonucleotides, including ones up to about 100 bases.

The oligonucleotides of this invention contain naturally occurringnucleotides or derivatives thereof. In some oligonucleotide embodimentsthe companion nucleotide residues contain pyrimidine nucleotidessubstituted at the 5 position with a carbon atom which is distally Pibonded to another atom as for instance 1-alkenyl, 1-alkynyl,heteroaromatic and 1-alkynyl-heteroaromatic groups such as5-(1-propynyl)-cytosine and -uridine nucleotides (see PCT PublicationNo. WO 93/10820 and U.S. Pat. No. 5,594,121). Other analogs of nativebases for use herein include alkylated purines or pyrimidines, acylatedpurines or pyrimidines, or other analogs of purine or pyrimidine basesand their aza and deaza analogs. These include, for example N⁴,N⁴-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N⁶-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyl uracil, inosine, N⁶ -isopentenyl-adenine,1-methyladenine, 2-methylguanine, 5-methylcytosine, N⁶ -methyladenine,7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil, pseudouracil,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-(1-propynyl)-4-thiouracil, 5-(1-propynyl)-2-thiouracil,5-(1-propynyl)-2-thiocytosine, 2-thiothymidine, and 2,6-diaminopurine.In addition to these base analogs, one can conveniently incorporate intothe invention oligonucleotides other base analogs, including pyrimidineanalogs including 6-azacytosine, 6-azathymidine, 5-trifluoromethyluracilor other bases previously described, see, e.g., bases, monomers oroligonucleotides described in WO 92/02258, WO 97/32888 and U.S. Pat. No.5,614,617.

Preferred bases include adenine, guanine, thymine, uracil, cytosine,5-methylcytosine, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and5-(1-butynyl)uracil, 5-(1-butynyl)cytosine.

Invention embodiments include the protected derivatives of native bases,their analogs and optionally protected monomer synthons containing suchbases, which one would typically use as intermediates to prepareinvention oligonucleotides (see, e.g., International Publication No. WO96/37504, U.S. Pat. No. 5,614,622 Iyer et al., "Nucleosides &Nucleotides", 14:1349-57, 1995, Uhlmann et al., "Chem Revs", 90:543-587,1990, S. Agrawal, Ed. Methods in Molecular Biology, Vol. 20, Protocolsfor Oligonucleotides and Analogs, pp. 165-189, Humana Press, 1993)

Embodiments of the oligonucleotides of the invention comprise a moietywhich is capable of effecting at least one covalent bond between theoligonucleotide and a nucleic acid duplex or strand. Multiple covalentbonds can also be formed by providing a multiplicity of suchcrosslinking moieties. The covalent bond is preferably to a base residuein the target strand, but can also be made with other portions of thetarget, including the saccharide or phosphodiester. Preferredcrosslinking moieties include acylating and alkylating agents, and, inparticular, those positioned relative to the sequencespecificity-conferring portion so as to permit reaction with the targetlocation in the strand. Exemplary crosslinking moieties are disclosedand claimed in PCT 91/03680. See also Praseuth ("Proc Natl Acad Sci"8:1349, 1988), Fedorova ("FEBS" 228:273, 1988), Meyer ("JACS" 111:8517,1989), Lee ("Biochemistry" 27:3197, 1988), Horne ("JACS" 112:2435,1990), Shaw ("JACS" 113:7765, 1991).

Invention embodiments include monomers and oligonucleotides containing1, 2, 3 or more invention bases and a 5' hydroxyl group protected with abase labile protecting group, including a dansylethoxycarbonyl group,which has been described, e.g., U.S. Pat. No. 5,631,362. Suchembodiments optionally include additional protecting groups.

Invention embodiments include monomers and oligonucleotides containing1, 2, 3 or more invention bases and an invention base or a non-inventionbase having an exocyclic nitrogen atom, where the nitrogen atom isprotected with a protecting group as previously described, e.g.,specification and claims 1, 2, 3, 4, 5, 6, 7 and 8 of U.S. Pat. No.5,623,068. Such embodiments optionally include additional protectinggroups.

Invention embodiments include optionally protected monomers andoptionally protected oligonucleotides containing 1, 2, 3 or moreinvention bases wherein the compositions possess N-branching, which hasbeen described, e.g., U.S. Pat. No. 5,623,049.

Invention embodiments include "hybrid" oligonucleotides, which contain1, 2, 3 or more invention bases and 2' modifications in one or tworegions or domains that comprise adjacent linked monomers, typicallyabout 2-8 linked monomers, usually about 2-3. One domain contains 2'modifications, while hydrogen is linked to the remaining monomers,typically about 4-10 adjacent linked monomers, usually about 6-8. Sucholigonucleotides contain at least one domain that is competent to serveas a RNase H substrate and comprises hydrogen at each 2' position andphosphodiester, phosphorothioate or phosphorodithioate 3',5' linkages.The other domain(s) contain a 2' modification(s), such as --O--(CH₂)₂ For --O--(CH₂)₂ --O--CH₃, that enhances binding affinity or nucleasestability. The 2'-modified domain(s) is usually not an efficient RNase Hsubstrate. The bases in hybrid oligonucleotides are the typical purinesand pyrimidines found in nucleic acids (G, A, T, C or U) or theiranalogs, which one finds in some oligonucleotide analogs (e.g., U.S.Pat. Nos. 5,484,908, 5,594,121 and 5,502,177, International PublicationNo. WO 93/10820). Intermediates used to prepare hybrid oligonucleotideswill typically contain appropriately protected derivatives of any bases.

Oligonucleotides of inverted polarity also fall within the scope of thisinvention. "Inverted polarity" means that the oligonucleotide containstandem sequences which have opposite polarity, i.e., one having polarity5'→3' followed by another with polarity 3'→5', or vice versa. Thesesequences thus are joined by linkages which can be thought of aseffectively a 3'--3' internucleoside junction (however the linkage isaccomplished), or effectively a 5'--5' internucleoside junction. For afurther description of suitable methods for making such oligonucleotidessee, e.g., WO 93/10820. Compositions of "parallel-stranded DNA" designedto form hairpins secured with AT linkages using either a 3'-3' inversionor a 5'-5' inversion have been synthesized by Van de Sande, "Science"241:551, 1988. In addition, oligonucleotides which contain 3'-3'linkages have been described (Horne, op cit; and Froehler,"Biochemistry" 31:1603, 1992). These oligonucleotides are useful asbinding partners for double stranded nucleic acids to form triple helix(or triplex) complexes as a means for detecting complementary sequencesand inhibiting of target gene expression (PCT 89/05769 and 91/09321).

Invention embodiments include polymers such as oligonucleotidescontaining 1, 2, 3 or more invention bases, where the polymer is acomponent of a complex or composition useful for transfecting thepolymer into a cell in vitro or in vivo. These complexes or compositionsare referred to herein as "transfection complexes". Such transfectioncomplexes optionally comprise one or more lipids, e.g., cationic oranionic lipids, as well as other lipophilic compounds such ascholesterol or colipids such as DOPE. The complexes optionally comprisean uncharged or a charged polymer such as polyethylene glycol, polybreneor a peptide, e.g., polylysine. The complexes optionally compriseunilamellar or multilamellar liposomes or vesicles.

As used herein, any compound(s), reagent(s) or treatment that enhancesdelivery of an invention oligonucleotide into a cell or tissue is a"permeation enhancing agent." Permeation enhancing agents are well knownand are usually present as transfection complexes containingoligonucleotides, e.g., unilamellar or multilamellar liposomes orvesicles. One uses permeation enhancing agents to prepare transfectioncomplexes containing invention oligonucleotides. The permeationenhancing agent are used in essentially the same manner as is used toprepared transfection complexes containing nucleic acids, non-inventionoligonucleotides or polymers into cells or tissues.

Invention transfection complexes optionally comprise an additionalnon-invention polymer(s), e.g., a nucleic acid expression vector(s), atherapeutic agent(s) (e.g., amphotericin B) or a peptide(s).

Invention transfection complexes comprising a lipid may be, as definedherein, "large", i.e., complexes having a maximum average dimension ofat least about 200 nm in length or diameter, typically having an averagelength or diameter of about 200-400 nm, occasionally having an averagelength or diameter of about 400-800 nm. Transfection complexescomprising a lipid may be "small", i.e., complexes having a maximumaverage dimension of about 15-200 nm in length or diameter, e.g., anaverage dimension of about 60-120 nm. Transfection complexes maycomprise a mixture of large and small complexes in about equalproportions or they may comprise a preponderance of small or largetransfection complexes, e.g., at least about 55%, or at least about60-80% of the complexes in a given preparation are large or small.

Transfection complexes comprising a lipid optionally include astabilizing compound(s), e.g., a monosaccharide or a disaccharide suchas glucose, trehalose, maltose or sucrose, that is present at the outersurface or at the inner surface or at both surfaces of transfectioncomplexes. Workers have described suitable compounds such as lipids,colipids and stabilizing compounds for making transfection complexes,methods to size the complexes and methods to use the complexes todeliver a polymer or monomer into the cytoplasm of a cell in vitro or invivo, e.g., U.S. Pat. Nos. 5,635,491, 5,633,156, 5,631,018, 5,629,184,5,627,159, 5,626,867, 5,620,689, 5,595,756, 5,543,152, 5,478,860,5,459,127, 5,264,618, 5,223,263, 5,194,654, 4,981,692, 5,077,056,4,522,803, 4,588,578, 4,885,172, 4,975,282, 5,059,421, 5,000,958,5,030,453 and 5,047,245, WO 96/01840, WO 96/01841, WO 97/30969, WO97/30732, Lewis, "Proc Natl Acad Sci" 93:3176-3181, 1996, U.S. patentapplication Ser. No. 08/672,206.

Invention transfection complexes useful for delivering the inventionoligonucleotides into cell cytoplasm also include complexes comprisinginorganic compounds, e.g., calcium phosphate.

R²¹

The R²¹ moiety is linked to invention oligonucleotides or monomersuseful for oligonucleotide synthesis. R²¹ is usually linked to the 2' orthe 3' position of furanose sugars. When R²¹ is a nuclease stabilityenhancing moiety, a broad range of structures may be used to increasestability of oligodeoxynucleotides or oligoribonucleotides containingphosphodiester linkages. Oligonucleotides having moieties other thanhydrogen or hydroxyl at the 2' position usually confer increasednuclease stability or increased binding affinity on the oligonucleotiderelative to hydrogen or hydroxyl. Enhanced nuclease stability isconveniently measured using dimers or short oligonucleotides asessentially described, e.g., WO 92/05186. One or two R²¹ moieties at the3' and 5' terminal monomers in an oligonucleotide will increasestability of the oligonucleotide to 3'- and 5'-exonucleases. One mayincrease an oligonucleotide's stability to endonucleases byincorporating R²¹ moieties that increase nuclease stability at internalmonomer positions.

In addition to increasing nuclease stability, some R²¹ moieties enhancebinding affinity of the oligonucleotide to which they are linked. Thesemoieties include fluorine and short unbranched optionally substitutedO-alkyl groups containing about 2-8 carbon atoms, where the alkyl groupis optionally substituted at the distal carbon atom with, e.g. --F, --OHor --NH₂, and optionally substituted with an ether at an internalcarbon, e.g., --O--(CH₂)₂ --O--(CH₂)₂ F, --O--(CH₂)₂ OCH₃ or --O--(CH₂)₂--O--(CH₂)₂ CH₃.

When R²¹ is a nuclease stability enhancing moiety, it will typicallycomprise --CH₃, ═O --NHR⁵ or a chain having a backbone containing 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 linked atoms, wherein the chain usuallycomprises carbon (C) atoms and optionally 1, 2, 3 or 4 atomsindependently selected from the group consisting of oxygen (O), nitrogen(N) and sulfur (S) atoms. The chain is usually linked to the sugarcarbon atom through --O--, --S--, --S(O)--, S(O)(O)--, --CH₂ --, ═CH--or --NH--. The chain is branched or unbranched, often it is unbranchedor has only limited branching, e.g., --CH₃, --CH₂ OH, --C₂ H₅ or --C₂ H₄OH. The R²¹ chain may comprise a C₂₋₁₂ alkyl group or a C₂₋₂₀substituted alkyl group. If R²¹ is a substituted alkyl group, usuallyonly 1, 2 or 3 carbon atoms are substituted. Suitable substituentsinclude those described above for substituted alkyl groups, e.g.,halogen (usually fluorine), --O-- or --OR⁵.

Invention embodiments include oligonucleotides and monomers where one ormore monomers comprise 2'-deoxyribose, ribose or arabinose sugars, ortheir carbocyclic analogs, having one or more 2' R²¹ modification suchas, --O-- alkyl, --NH--alkyl, --S--alkyl, --OR⁵, --NHR⁵, --SR⁵, -halo(usually --F), --R⁴⁴ -alkyl or --R44-substituted alkyl wherein the alkylor substituted alkyl group usually comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 carbon atoms, usually about 2-6 carbon atoms, where R⁴⁴ isindependently --O--, --S--, --NH-- or --CH₂ --, usually --O--. Theoligonucleotide linkages connecting such monomers are 3',5' linkages.The alkyl groups at the 2' position typically have 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12 carbon atoms which are optionally present asmethylene groups (--CH₂ --) and optionally have 1, 2, 3 or 4 ether(--O--) or other substitutions, e.g., O-alkoxyalkyl (C₂ -C₁₂ alkyl),--O--(CH₂)₂₋₈ --CH₂ CO₂ H, --O--(CH₂)₂₋₈ --CH₂ N(R⁵)₂, --(--O--(CH₂)₂₋₄--O--(CH₂)₂₋₄ --O--(CH₂)₂₋₄ --R⁶⁵), --O(CH₂ CH₂ O)_(r) CH₂ --R⁶⁵,--O--CH₂ CH₂ --R⁶⁵, --O(CH₂ CH₂)O(CH₂ CH₂)R⁶⁵, --OCH₂ CF₂ CF₃, where R⁶⁵is --H, halo (usually fluorine), --OR⁵, --OCH₃, --NHR⁵, --SR⁵, and r is1, 2, 3 or 4, usually 1 or 2.

The alkyl groups at the 2' position also include substituted alkyl,e.g., --O-- alkylamino, --S-alkylamino, --NH-alkylamino, or theirprotected derivatives, wherein the alkyl group contains 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 carbon atoms, which are all optionally present asmethylene carbons (--CH₂ --). Usually the alkyl group or substitutedalkyl group at R²¹ will contain 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.Such groups include --O-methyl, --O-ethyl, --O-n-propyl, --O-allyl,--O--(CH₂)₂₋₆ OH, including --O--(CH₂)₂ OH, --O--(CH₂)₂ F, --O--CH₂CHF₂, --O--CH₂ CF₃, --O--(CH₂)₂₋₆ OCH₃, including --O--(CH₂)₂ OCH₃,--O--(CH₂)₂ OCH₂ CH₃, --O--(CH₂)₂ OCH₂ CH₂ OH, --O--(CH₂)₂ OCH₂ CH₂ F,--O--(CH₂)₂ NHR⁵, --O--(CH₂)₃ NHR⁵, --O--(CH₂)₄ NHR⁵, --O--(CH₂)₂ F,--O--(CH₂)₃ F, --O--(CH₂)₄ F, --O--CH₂ --CF₂ CF₃, --O--(CH₂)_(S) R⁶⁵,--O--(CH₂)₂ --[O--(CH₂)₂ ]_(r) R⁶⁵, --NH-methyl, --NH-ethyl,--NH-n-propyl, --NH--(CH₂)₂ OH, --NH--(CH₂)₃ OH, --NH--(CH₂)₂ F,--NH--CH₂ --CF₂ CF₃, --NH--(CH₂)_(s) R⁶⁵, --S-methyl, --S-ethyl,--S-n-propyl, --S-allyl, --S--(CH₂)_(s) OH, --S--(CH₂)₃ OH --S--(CH₂)₂ Fand --S--(CH₂)_(s) --[O--(CH₂)₂ ]_(r) R⁶⁵, where s is 2, 3, 4, 5, 6, 7or 8. R²¹ moieties do not include unstable species at the 2' position,e.g., --O--O-- or --S--O--.

Other suitable R²¹ at the 2' or 3' position of optionally protectedinvention monomers or optionally protected invention oligonucleotidesinclude --² H, --³ H, --NHOR⁵⁵, ═NH, --N₃, --CN, --CH₂ CN, --CHCl₂,--CFH₂, --CF₂ H, ═CH₂, ═CF₂, --CH₂ CH═CH₂, ═O, ═CHC(O)OR⁵⁵ (including═CHC(O)OCH₃ and ═CHC(O)OCH₂ CH₃), --OC(S)OC₆ H₅, t-butyldimethylsilylether, triisopropylsilyl ether, a 2' amino group protected by anN-phthaloyl protecting group or a fluorescent label, R⁵⁵ isindependently R⁵, alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbonatoms or substituted alkyl having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbonatoms where the carbon atoms in R⁵⁵ are optionally all present asmethylene (--CH₂ --) or substituted methylene (--CH(substitution)-)moieties. Workers have described suitable 2' modified monomers andoligonucleotides, e.g., WO 97/14706, WO 96/05298, WO 93/13121, and WO91/06556 and U. S. Pat. Nos. 5,631,360, 5,627,053, 5,623,065, 5,576,302and 5,578,718.

Methods for Synthesis

The compounds of structure (1) where R¹ is a linker or H are prepared bymethods known in the art per se and as more fully described below.Typically, such compounds are prepared from a 5-bromouracil,5-bromouridin-1-yl, 5-iodouracil, 5-iodouridin-1-yl substitutedderivative as shown in the synthetic schemes below and subsequentreactions close the polycyclic ring. In these embodiments the hydroxyl,amino and any other labile groups of R¹ are protected as required. Inanother approach, R¹ of the starting material is H or a protecting groupand one adds the linker after the ring closure steps set forth in theschemes, in the same fashion as has heretofore been employed in thealkylation of pyrimidine bases intended for use as antiviral compounds.

In those embodiments in which R¹ is a binding partner such as a polymerthe compounds of this invention are synthesized by covalentlycrosslinking the linker modified polycyclic base of this invention tothe binding partner, or (where the binding partner is a polymer) byincorporating into the polymer a monomer unit which is substituted by aninvention polycyclic base.

In the first embodiment (polymer grafting) a R¹ -substituted polycyclicsubstructure is covalently bonded via any conventional cross-linkingagent to the polymer. Most conveniently, structure (1) compounds inwhich R¹ is hydroxyl- or amino-substituted alkyl are readilycross-linked to reactive groups present in the molecule to be labeled asnoted above. Exemplary cross-linking agents include succinic anhydride,N-hydroxysuccinimide esters (biotin NHS ester), epoxides,isothiocyanates, imidates, DCC (dicclohexylcarbodiimide), EDC(1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide), BOP, andglutaraldehyde, see, e.g., EP 0 063 879, Ruth "J Org Chem" 43:2870,1978, Bergstrom, JACS 100:8106, 1978, Bigge, JACS 102:2033, 1980.Cyanogen bromide activated carbohydrates also are used. Thecross-linking agents are used to bond the R¹ -substituted polycycle tothe polymer in the same fashion as polymers heretofore have beencross-linked to ligands, e.g., to hydroxyl or amino-bearing moieties. Anexample of a suitable method is described per se in Cook et al., U.S.Pat. No. 5,218,105. This method is readily applied to covalently bond anamino-substituted R¹ linker to the 5' terminus of an oligonucleotide.

When R¹ or R² are amino substituted, the following exemplary syntheticapproaches are suitable for crosslinking amines with other moieties:

--CH₂ NH₂ +R¹⁰ --C(N⁺ H₂)--OR¹⁰ →--CH₂ NHC(N⁺ H₂)--R¹⁰

--CH₂ NH₂ +R¹⁰ --N═C═S→--CH₂ NHC(S)NH--R¹⁰

--CH₂ NH₂ +R¹⁰ -epoxide→--CH₂ NHCH₂ CH(OH)--R¹⁰

where R¹⁰ is an organic moiety optionally containing a hapten or otherdetectable moiety such as biotin or avadin.

In the second embodiment (copolymerization) the R¹ linker is capable offunctioning as a monomer for copolymerization with other monomer unitsthat may or may not be substituted with the polycyclic substructure ofstructure (1). In some embodiments, the R¹ linker is an alkylcarboxylate, an alkyl amine or an amino acid for incorporation intopeptides by in vitro methods. However, in the typical embodiment the R¹polymeric binding partner is an oligonucleotide as depicted in structure(2), and these conveniently are made by copolymerization with anucleotide analog substituted with the polycyclic substructure. Thestarting materials for the synthesis of structure (2) generally arecompounds of structure (1) in which R¹ is ribose or deoxyribosesubstituted with appropriate protecting and coupling groups furtherdescribed above. Suitable starting monomers for oligonucleotides havingphosphodiester substitute linkages are set forth in Table 1, and theyare prepared in the same fashion as other nucleotide analog basesdescribed in the literature. Similarly, conventional phosphodiester orphosphorothioate linkages are prepared from nucleotide analogscontaining coupling groups D and D¹ described above. The compounds ofthis invention then are incorporated into the desired oligonucleotide byknown methods of in vitro synthesis described in the referenced methods.Alternatively, polycylic substructure-substituted nucleotidetriphosphates may be incorporated into oligonucleotides as cytosineanalogs by DNA polymerase or reverse transcriptase in vivo or in vitro(see Ward, U.S. Pat. No. 4,711,955). In this case, R¹ is ribosyl ordeoxribosyl triphosphate, or a triphosphorylated analog thereofrecognized by DNA polymerase or reverse transcriptase which is thenincorporated into an oligonucleotide by template-directed transcription.

Synthesis of oligonucleotides containing 3 or more nucleotide residuesis optionally accomplished using synthons such as dimers (which containsubstitute or diester linkages) or trimers, each carrying a terminalcoupling group suitable for use with amidite, H-phosphonate or triesterchemistries. The synthon is then linked to the oligonucleotide oranother synthon via a phosphodiester or phosphorous-containingphosphodiester substitute linkage.

Oligonucleotides containing phosphorothioate, methylphosphonate andphosphodiester linkages are readily prepared by solid-phaseoligonucleotide synthesis techniques. A description of modificationsuseful in the synthesis of phosphorothioate linked oligonucleotides arefound, for example, in EP 288,163, wherein the oxidation step in solidphase automated synthesis using amidite chemistry can be independentlyadjusted at any step to obtain the phosphorothioate. An alternate methodfor synthesis of oligonucleotides with phosphorothioate linkages, viahydrogen phosphonate chemistry, has also been described (Froehler "NAR"14:5399, 1986). Sulfurization is accomplished using reagents such astetraethylthiuram disulfide, dibenzoyl tetrasulfide, thiophosphoric aciddisulfide, 3H-1,2-benzodithiol-3-one 1,1-dioxide and the like asdescribed (Vu, "Tet Lett" 26:3005, 1991; Rao, "Tet Lett" 33:4839, 1992;U.S. Pat. No. 5,151,510; Iyer, "JOC" 55:4693, 1990; Dahl, "SulfurReports" 11:167, 1991). These sulfurization reagents are used witheither phosphoramidite or hydrogen-phosphonate chemistries. Synthesis ofphosphorothioate oligonucleotides having controlled stereochemistry isused to generate stereoregular invention oligonucleotides as described(EP 506,242). Thionomethyl phosphonate is prepared withmethylphosphonamidite followed by sulfurization as described (Roelen,"Tet Lett" 33:2357, 1992) or with the sulfurization reagents describedabove.

One prepares various structure (1) compounds as described below and inthe examples. ##STR17##

Scheme A depicts preparation of structure (1) compounds where R⁴⁷ is--O-- or --S--; and R^(2A) --OH is R² which has a free hydroxyl.

Step one is conducted by heating the reaction mixture containing (100)in an organic solvent to at least about 50° C., generally for about 3-4hours. Step two is performed by reacting (102) in an organic solvent forabout 6-48 hours, generally for about 10-20 hours at about 15° C. toreflux temperature, generally at about 18-25° C. The R² moiety is linkedunder Mitsunobu conditions to (103) in step 3 by reacting about 1-1.5equivalents of the alcohol, i.e., R^(2A) --OH, using an activating agentas a leaving group, such as triphenylphosphine (Ph₃ P) and diethyldiazocarboxylate (DEAD) to obtain (104). In step 4, (105) is prepared byforming the ring containing R⁴⁷ by (1) incubating (104) in a polarorganic solvent, typically an alkanol containing 1, 2, 3, 4, 5 or 6carbon atoms, such as methanol or ethanol, containing a mild base suchas NH₃, TEA (triethylamine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) or(2) refluxing in ethanol in the presence of potassium fluoride.Generally (104) is incubated in saturated NH₃ in methanol for about 2-3days to afford (105).

The use of (102) in which one R⁴⁷ is --O-- and the other is --S-- willproduce a mixture. One optionally isolates each (104) component or oneoptionally converts the (104) mixture to a (105) mixture. One optionallyseparates the mixtures at any convenient point by standard methods,e.g., silica gel chromatography, or HPLC.

When one prepares compounds according to scheme A and R¹ is amonosaccharide, e.g., 2'-deoxyribose, 2'-deoxy-2'-R²¹ -substitutedribose or arabinose, the sugar hydroxyls in (100) are usually protected,generally using base-labile protecting groups, e.g., acetate,proprionate, butyrate, phenoxyacetyl. The step 4 reaction under basicconditions removes the protecting groups, which facilitates the ringformation reaction resulting in (105). When R¹ is not a monosaccharide,it is generally a protecting group or an optionally protected linker,e.g., --(R⁴⁸)_(X) --R⁴⁹, where R⁴⁸ is independently --CH₂ --, --O--,--S--, --NH-- or --C(O)--, X is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R⁴⁹usually is a functional group suitable for linking to a solid support,monomer or polymer, including --OR⁵, --SR⁵, --N(R⁵)₂, --C(O)N(R⁵)₂,--NR⁵ C(O)H, --C(O)H and --C(O)OR⁵, R⁵ is independently --H, aprotecting group, or both R⁵ together are a protecting group. Generallythe R⁴⁸ group that is adjacent to R⁴⁹ is ethylene (--CH₂ --CH₂ --) andgenerally only 0, 1 or 2 R⁴⁸ are moieties other than --CH₂ --, e.g., oneR⁴⁸ is --O-- or --C(O)-- and the remaining R⁴⁷ are all --CH₂ --.Exemplary compounds where R¹ is a monosaccharide have the structures(130)-(134), which correspond to compounds (100)-(101) and (103)-(105)respectively. For compounds (130)-(132), R⁵ at sugar hydroxyls istypically a base labile protecting group such as acetyl and R³⁷ istypically --O--. During conversion of (133) to (134), the R⁵ protectinggroup is removed from the sugar under ring closure reaction conditions,which facilitates ring closure. ##STR18##

Structure (134) compounds may be converted to monomers suitable foroligonucleotide synthesis. Such monomers typically have a coupling groupat the 3' position, e.g., H-phosphonate, or a phosphoramidite such as aβ-cyanoethylphosphoramidite, N,N-diisopropylamino-β-cyanoethoxyphosphineor N,N-diisopropylaminomethoxyphosphine. The 5' position will contain aDMT-O-- or other protecting group suitable for oligonucleotidesynthesis. The monomers may alternatively have a coupling group at the5' position and a protecting group at the 3' position. The protectingand coupling groups are added sequentially. Scheme B shows synthesis ofstructure (1) compounds where R⁶ in R² is --CH₂ --. In scheme B, Y is 1,2, 3 or 4; R⁵⁰ is independently --CH₂ --, --C(O)--, --(CH₂)₂ --O--(CH₂)₂--, --(CH₂)₂ --NR⁵ --(CH₂)₂ --, --(CH₂)₂ --S--(CH₂)₂ --, --CH(N(R⁵)₂)--,--CH(COOR⁵)-- or --C(CH₃)--, --C(C₂ H₅)-- but adjacent moieties are notC(O), usually R⁵⁰ is --CH₂ --; TFA is trifluoroacetate; and CBZ iscarboxybenzoyl; and (96) is HC.tbd.C(R⁵⁰)_(y) --NH-TFA. Protectinggroups present in R⁵⁰ are stable to the reaction conditions shown.##STR19##

Compound (97) is converted to (98) by hydrogenation reaction in alcohol,usually methanol or ethanol, at about 15-25° C. for about 10-24 hours,usually about 12-18 hours. The catalyst is removed and the filtrate isconcentrated.

Compound (98) in trimethyl orthoformate is converted to (99) in thepresence of acid, e.g., methanesulfonic acid at about 15-25° C., usuallyabout 18-22° C. for about 20-120 minutes. The reaction is cooled andquenched with a base, e.g., an organic base such as TEA. The reactionmixture is concentrated and purified, e.g., by flash columnchromatography on silica gel.

Compound (106) is prepared by reacting (99) in organic solution such asDMF, CH₂ Cl₂ or CH₂ Cl₂ /DMF (about 2:1 v/v) with K₂ CO₃ at about 15-25°C. for about 30 minutes, followed by addingphenyltrifluoromethanesulfonimide and stirring the mixture for about10-24 hours, usually about 12-16 hours. The reaction mixture is thendiluted with CH₂ Cl₂, washed with water once or twice and concentratedand purified.

Compound (96) is prepared by reacting HC.tbd.--C(R⁵⁰)_(Y) NH₂ with ethyltrifluoroacetate at about 15-25° C. for about 10-24 hours, washing withsaturated aqueous NaHCO₃ and concentrated. Compound (96) is purified bydistillation.

Compound (107) is prepared by stirring an organic solvent such as DMFcontaining about 2 equivalents of (96), about 2 equivalents of anorganic base such as TEA, and Pd((PPh)₃)₄, CuI and about 1 equivalent of(106) at about 15-25° C. for about 18-36 hours. The organic phase iswashed with water, dried and purified by silica gel chromatography, toobtain (107).

Compound (107) is hydrogenated in ethanol in the presence of 10% Pd/C atroom temperature. The catalyst is filtered off, the filtrate isconcentrated and then treated with conc. NH₄ OH:dioxane (1:1) to afford(108). The amino group in (108) was protected with CBZ to afford (109).

Compound (110) is prepared by treating (109) in ethanol with aqueous HCl(about 3 N) at about 15-45° C., usually about 40° C., for about 30-120minutes, usually about 60 minutes. The product is dried and optionallyazeotroped using e.g., CH₃ CN, several times.

Compound (111) is prepared by reaction of (100) with mesitylenesulfonylchloride in the presence of a tertiary amine such as TEA.

Compound (112) is prepared by stirring a mixture of (110) and (111) inorganic solution containing about 2 equivalents of an organic base suchas DBU or TEA at about 15-25° C. for about 10-24 hours. The reactionmixture is washed with an aqueous 10% citric acid solution, dried andpurified by silica gel chromatography.

Compound (113) is prepared by treating (112) with saturated NH₃ inmethanol at about 15-25° C. for about 3-4 days. The reaction mixture isdried, concentrated and purified by silica gel chromatography.

Compound (114) is prepared by hydrogenation of (113) in the presence of10% Pd/C at about 15-25° C. for about 3-6 hours. Catalyst is removed,washed, and the filtrate is concentrated to dryness. The amino group in(114) is protected with FMOC to afford (115).

Where R¹ in scheme B is an optionally protected monosaccharide such as2'-deoxyribose, 2'-deoxy-2'-R²¹ -substituted ribose, 2'-deoxy-2'-R²¹-substituted arabinose, ribose or arabinose, the sugar's hydroxyl groupsin compounds (111) and (112) are usually protected with a base-labileprotecting group such as acetyl, propionyl and phenoxyacetyl. Theseprotecting groups are removed by treatment with base during synthesis of(113).

Exemplary compounds where R¹ is a monosaccharide have the structures(135)-(139), which correspond to compounds (111)-(115) respectively. Forcompounds (135)-(136), R⁵ at sugar hydroxyls is typically a base labileprotecting group such as acetyl. R³⁷ is typically --O--. Duringconversion of (136) to (137), the R⁵ protecting group is removed fromthe sugar under ring closure reaction conditions, which facilitates ringclosure.

Structure (139) compounds may be converted to monomers suitable foroligonucleotide synthesis. Such monomers typically have a coupling groupat the 3' position, e.g., H-phosphonate, or a phosphoramidite such as aβ-cyanoethylphosphoramidite,N,N-diisopropyl-amino-β-cyanoethoxyphosphine orN,N-diisopropylaminomethoxyphosphine. The 5' position will contain aDMT-O- or other protecting group suitable for oligonucleotide synthesis.The monomers may alternatively have a coupling group at the 5' positionand a protecting group at the 3' position. The protecting and couplinggroups are added sequentially. ##STR20##

Scheme C shows synthesis of structure (1) compounds where R⁶ in R² is--NH--. ##STR21##

In scheme C, R⁵² is --(CHR^(52A))--(R^(52B))--CHR^(52A) --, whereR^(52A) is --H or C₁ -C₆ alkyl (typically C₁ -C₃), usually --H, andR^(52B) is a bond, --CHR^(52A) --O--CHR^(52A) --, --CHR^(52A)--S--CHR^(52A) --, --CHR^(52A) --NR⁵ --CHR^(52A) --, C₁ -C₁₀ alkylene(typically C₃ -C₆) optionally substituted with 1 or 2 moieties selectedfrom the group consisting of C₁ -C₆ alkyl (typically C₁ -C₃ alkyl),--OR⁵, ═O, --NO₂, --N₃, --CN, --COOR⁵, or --N(R⁵)₂. In R⁵², anyheteroatom in the spacer chain will be separated from the nitrogen atomsthat R⁵² is linked to by one methylene and one or more --CHR^(52A) --moieties. Typically, adjacent carbon atoms in R^(52B) are notsubstituted with --OR⁵, ═O, --NO₂, --N₃, --CN, --COOR⁵, or --N(R⁵)₂.Protecting groups present on (119A) are usually stable to oxidizingconditions and labile to basic conditions. The protected intermediate(119A) is prepared by reacting phthalic anhydride with the appropriateHOCH--R⁵² --NH₂ moiety to yield a phthalimide compound or by reacting aphthalimide compound with HOCH--R⁵² --Br.

Compound (117) is prepared by reacting (101) or (111) with about 1.0-1.5equivalents, usually about 1.1 equivalents of (116) and about 1-2equivalents, usually about 2 equivalents of organic base such as DBU orTEA in an organic solvent such as CH₂ Cl₂ /CCl₄ (about 1:1) or CH₂ Cl₂by reaction at about 10-30° C., usually at about 18-25° C. for about16-48 hours, usually about 20-28 hours.

Where R¹ in scheme C is an optionally protected monosaccharide such as2'-deoxyribose, 2'-deoxy-2'-R²¹ -substituted ribose, 2'-deoxy-2'-R²¹-substituted arabinose, ribose or arabinose, the sugar's hydroxyl groupsin compounds (101), (111) and (117) are usually protected with abase-labile protecting group such as acetyl, propionyl andphenoxyacetyl. These protecting groups are removed by treatment withbase during synthesis of (118). Compound (118) was hydrogenated inethanol and acid to obtain (119). Compound (120) was obtained byreductive alkylation of (119) with aldehydes.

Compound (120) is optionally converted, without removing the nitrogenprotecting group linked to the exocyclic amine, to a monomer suitablefor use in oligonucleotide synthesis, e.g., (121) (not shown), byprotecting the 5' hydroxyl group by reaction with a protecting groupreagent such as DMT-Cl (4,4'-dimethoxytrityl chloride) to obtain the5'-protected derivative (not shown). Compound (121) is then converted toa derivative (121A) (not shown) suitable for coupling the 3' hydroxylgroup with another 5' hydroxyl group, i.e., a coupling group such asH-phosphonate or a phosphoramidite, e.g.,N,N-diisopropylamino-β-cyanoethoxyphosphine orN,N-diisopropylaminomethoxyphosphine, is linked to the 3' position.Exemplary compounds where R¹ is a monosaccharide have the structures(140)-(143), which correspond to compounds (117)-(120) respectively. Forcompounds (140)-(141), R⁵ at sugar hydroxyls is typically a base labileprotecting group such as acetyl and R³⁷ is typically --O--. In structure(143) compounds, the 5' or 3' oxygen, usually the 3' oxygen, is linkedto --H or a coupling group such as H-phosphonate, or a phosphoramiditesuch as β-cyanoethylphosphoramidite,N,N-diisopropylamino-β-cyanoethoxyphosphine orN,N-diisopropylaminomethoxyphosphine. During conversion of compound(140) to (141), the R⁵ protecting group is removed from the sugar underring closure reaction conditions, which facilitates ring closure.##STR22##

Structure (143) compounds may be converted to monomers suitable foroligonucleotide synthesis. Such monomers typically have a coupling groupat the 3' position, e.g., H-phosphonate, or a phosphoramidite such as aβ-cyanoethylphosphoramidite, N,N-diisopropylamino-β-cyanoethoxyphosphineor N,N-diisopropylaminomethoxyphosphine. The 5' position will contain aDMT, MMT or other protecting group suitable for oligonucleotidesynthesis. The monomers may alternatively have a coupling group at the5' position and a protecting group at the 3' position. The protectingand coupling groups are added sequentially. During oligonucleotidesynthesis, (143) is incorporated into an oligonucleotide such, as one ofstructure (2), by standard methods and the phthalamide protecting group,along with other base labile protecting groups at R⁵² are removed usingbasic conditions, e.g., NH₄ OH or NH₂ CH₃, to yield deprotected orpartially deprotected --NH--R⁵² --NH₂ as R².

Straightforward variations of schemes A-C can be used to prepare otherstructure (1) compounds. For example, scheme D depicts a method toprepare structure (1) compounds where R² comprises a cytosinederivative. In scheme D, iPr is isopropyl; R⁵⁹ is a portion of an R²moiety having the structure --R⁶ --R⁶⁰ --, where R⁶ is usually --O--,--S--, --NH-- or --CH₂ --; R⁶⁰ is --(CH₂)_(Z3) --(R⁶¹)_(Z1) --(CH₂)_(Z2)--; R⁶¹ is --O--, --S--, --NR⁵ --, --C(O)--, --CH₂ --O--CH₂ --, --CH₂--NR⁵ --CH₂ -- or --CH₂ --S--CH₂ --; Z3 is 1, 2 or 3, usually 1; Z1 is 0or 1, usually 0; and Z2 is 1, 2 or 3, usually 1; any functional groups,e.g., --OH, --NH₂, --COOH, --SH, that are optionally present at R²¹ areprotected. Compound (124) is 3',5'-diacetyl-O⁴-sulfonyl-2'-deoxyuridine, the synthesis of which is described in theexamples. ##STR23##

Compound (126) of scheme D is converted to a monomer suitable foroligonucleotide synthesis using standard methods, e.g., treatment of(126) with a protecting group such as DMT-Cl protects the 5' oxygen atomand yields compound (127) (not shown). One then links a coupling groupsuch as H-phosphonate or a phosphoramidite group such asN,N-diisopropylamino-β-cyanoethoxyphosphine orN,N-diisopropylaminomethoxyphosphine to the 3' hydroxyl group, of (127)to obtain (128) (not shown), which is suitable for oligonucleotidesynthesis. Variation of the synthesis shown in scheme D is used toprepare compounds of structure (129) or (129A). ##STR24##

Similarly, structure (1) compounds where R² is ##STR25## are synthesizedusing variations of scheme D and the corresponding intermediates, e.g.,reaction of (123) with 2-halopyridine (Bernatowicz "JOC" 57:2497 1992).

Compounds of structure (1) where R² is ##STR26## where R²⁹ is --N--,--CH-- or --C(CH₃)--, R⁶² is --H, --NH₂ or --NH(CH₃) and R⁶ is --O-- or--S-- are synthesized using scheme A, while scheme B is used when R⁶ is--CH₂ -- and scheme C is used when R⁶ is --NH--.

Compounds of structure (1) where R² is ##STR27## where R⁶¹ is --H, alkylhaving 1, 2, 3 or 4 carbon atoms or optionally protected substitutedalkyl having 1, 2, 3, 4, 5 or 6 carbon atoms including --CH₃ and --CH₂CH₃, and R⁶ is --O-- or --S-- are synthesized using scheme A, whilescheme B is used when R⁶ is --CH₂ -- and scheme C is used when R⁶ is--NH--.

Compounds of structure (1) where R² is --R⁶ --R⁶⁰ --N(R₃)₂, including--R⁶ --(CH₂)_(t) --N(R₃)₂, and R⁶ is --O-- or --S-- are synthesizedusing scheme A, while scheme B is used when R⁶ is --CH₂ -- and scheme Cis used when R⁶ is --NH--.

Compounds of structure (1) where R² is ##STR28## including ##STR29## andR⁶ is --O-- or --S-- are synthesized using scheme A, while scheme B isused when R⁶ is --CH₂ -- and scheme C is used when R⁶ is --NH--.Compounds containing these structures where R⁶ is --S-- or --O--, ingeneral are synthesized using Scheme A and compound (103), e.g., using#1154-093 described in the examples below, and the correspondingprotected alcohols. Compounds where R⁶ is --CH₂ -- are obtained byfollowing Scheme B. Compounds where R⁶ is --NH-- can be obtained usingcompound (119), e.g., using #1090-68 and protected aldehydes describedin the examples below, by following Scheme C.

Schemes E and F illustrate the synthesis of alcohols containing animidazole moiety (see, e.g., Munk "J. Med. Chem." 40:18 1997). In schemeE, R⁶² is R⁵ or alkyl having 1, 2, 3, or 4 carbon atoms and R⁶³ is--OR⁵, --(CH₂)_(Z3) OR⁵ or --(CH₂)₁₋₂ --R⁴⁷ --(CH₂)₁₋₂ --OR⁵ and Z4 is0, 1, 2, 3, 4 or 5. In scheme F, Z5 is 1, 2, 3, 4 or 5. ##STR30##

Scheme D is also used to obtain compounds of structure (1) where R²comprises, for example, a moiety such as --(CH₂)₂₋₄ --NH((CH₂)₂₋₄ NH)₀₋₄--(CH₂)₂₋₄ NH₂, --(CH₂)₂₋₄ NH(CH₂)₂₋₄ N((CH₂)₀₋₄ --NH₂)₂, or when R² is--R⁶ (CH₂)_(t) N(R³)₂ and one R³ is H, CH₃, CH₂ CH₃, a protecting groupor --(CH₂)_(v) --N(R³³)₂ and the other R³ is --(CH₂)_(v) --N(R³³)₂,--CH(N[R³³ ]₂)--N(R³³)₂, (48), (49) or (50).

Converting a compound of structure (4) to a compound of structure (1) isaccomplished by a method comprising displacing R²⁴. In this method, R²⁴usually is --Br and R²⁷ is usually --CH--.

R² groups containing a diol, e.g. (CH₂ OH)₂ --CH--(CH₂)_(n) --O-- wheren is 1-8, are converted by cleavage to the aldehyde CHO--CH₂ --(CH₂)_(n)--O-- using sodium periodate. Reductive alkylation of the aldehyde to aprimary or secondary amine is accomplished using N(R^(3A))₂ where R^(3A)independently is --H, C₁₋₆ alkyl, including --CH₃, --CH₂ CH₃ or aprotecting group, usually --H or --CH₃.

To the extent that a compound within the claims' scope can not bedirectly synthesized using the schemes above or the examples below, theartisan will employ straightforward methods known for preparing suchcompounds, see e.g., B. M. Trost & I. Fleming, eds. "ComprehensiveOrganic Synthesis", volumes 1-8, Pergamon Press; M. Fieser, ed. "Fieserand Fieser's Reagents for Organic Synthesis", volumes 1-17, John Wiley &Sons; and A. F. Finch Ed. "Theilheimer's Synthetic Methods of OrganicChemistry", volumes 1-49, latest editions, S. Karger AG.

Uses for the Compounds of this Invention

The compounds of this invention find uses in the diagnostic, analyticand therapeutic fields, or as intermediates in the preparation ofcompounds useful in such fields.

The R² -substituted compounds of structure (1) are useful asintermediates in the preparation of the labeled biopolymers of structure(1), wherein a biopolymer is rendered fluorescent or otherwisedetectably labeled by linkage to the polycyclic substructure. It is mostconvenient, however, to use the appropriate structure (1) compounds asmonomers in the preparation of nucleic acids or oligonucleotides. Thelabeled biopolymers are employed in diagnostic assays or preparativeprocedures in the same fashion as other fluorophor-labeled biopolymers,e.g. in fluorescence polarization methods, fluorescence activated cellsorting, competitive-type EMIT immunoassays and the like.

The linker- and hydrogen-substituted compounds of structure (1) areuseful as intermediates to prepare materials suitable for use inaffinity purification of guanine or guanine-containing compounds, e.g.,nucleosides. The structure (1) compounds form hydrogen bonds withguanine and one can thus link the structure (1) base structure to anappropriate support material or polymer to prepare an affinity resinsuitable for binding to guanine-containing compounds, e.g., nucleosides,nucleotides and oligonucleotides or similar compounds containing guanineanalogs, e.g., 7-deazaguanine. Typical linker-derivatized structure (1)compounds optionally contain a linker of structure --(R⁴⁸)_(X) --R⁴⁹,defined above in the discussion under Scheme A.

The monomers are of particular use in preparing oligonucleotides fordiagnostic or therapeutic use. Since oligonucleotides having 2 or morenucleotides or nucleotide analogs bearing the polycyclic substructurewill usually exhibit greatly increased Tm, such oligonucleotides areparticularly useful in therapeutic or diagnostic utilities where highlystable duplex hybridization structures are desired. Since theseoligonucleotides frequently are fluorescent, changes in theoligonucleotide fluorescence can be followed as the oligonucleotidebinds to complementary nucleic acid or oligonucleotide sequences. Thesechanges are detectable as modifications in energy transfer, e.g.,fluorescence quenching or shifts in activation or emissionwavelength(s).

The polycyclic substructure labeled oligonucleotides are employed indiagnostic or analytic methods in the same fashion as other labeledoligonucleotides. For example, the oligonucleotides are used inhybridization methods in which an antibody capable of bindingbase-paired structure (1) is used to detect binding of theoligonucleotide to a target nucleic acid sequence. In addition, changesin fluorescent character can be assayed as described above. Typically,2, 3 or more polycyclic substructure labeled oligonucleotides are usedin a hybridization assay. One oligonucleotide is labeled at its 3' endwith a polycyclic substructure containing nucleotide while the othernucleotide is labeled at its 5' end with the same or another polycyclicsubstructure or with a different fluorophor such as fluorescein orrhodamine capable of energy transfer. The two oligonucleotides recognizea complementary sequence in which the 3' end of the target sequencebinds the oligonucleotide bearing the 3'-terminal fluorophor and theadjacent 5' sequence of the target binds to the oligonucleotide bearingthe 5' terminal fluorophor. Binding is assayed by measuring a change influorescence of either or both of the oligonucleotides when they bind intandem according to the illustrated model. In other embodiments only asingle labeled oligonucleotide is employed in the hybridization method.The oligonucleotides of this invention thus are useful in solution phasehybridization diagnostics, i.e., it is not necessary to perform a phaseseparation in order to detect labeled oligonucleotide binding.

Detecting a target base sequence in a nucleic acid or an oligonucleotideusing an invention oligonucleotide is accomplished by a methodcomprising (i) mixing or contacting a sample suspected of containing anucleic acid with an optionally labeled invention oligonucleotidecomprising at least about 7 bases, usually about 12-30 bases, where theprotecting groups have been removed, (ii) allowing time sufficient forthe invention oligonucleotide to bind to the target base sequence, (iii)separating unbound invention oligonucleotides from bound inventionoligonucleotides and (iii) detecting the presence, absence or amount ofbound invention oligonucleotide. Aspects of the invention includeconducting any one of these steps individually, which are each part ofthe complete method. The use of known hybridization methods andconditions are applied to accomplish the method.

In this method to detect target base sequences, one may optionally useinvention oligonucleotide and target base sequences that aresubstantially complementary to each other, i.e., sequences having 1 orno mismatches per about every 15-30 bases. The target base sequences tobe detected are generally present in a cell, in a cell or tissue extractor, usually, in a purified nucleic acid or oligonucleotide preparation,e.g., a sequence encoding a portion of a cytokine, cell surfacemolecule, an enzyme such as farnesyl protein transferase or an oncogenesuch as neu, myc, raf, ras or c-ras. The target base sequences to bedetected are generally present in RNA or single stranded DNA, althoughthe invention oligonucleotides are also useful to detect base sequencesin duplex nucleic acids.

Another invention embodiment is a method comprising incubating a cellwith a deprotected invention oligonucleotide containing at least about 7bases, usually about 12-30 bases, wherein the invention oligonucleotideis optionally present in a transfection complex comprising the inventionoligonucleotide and a permeation enhancing agent. This method is used tointroduce optionally labeled invention oligonucleotides into cellcytoplasm or vacuoles.

One optionally conducts this method using invention oligonucleotideshaving a octanol:water partition coefficient of about -0.5 to about 2.5,typically about 0.0 to about 2.0, usually about 0.5-1.5, and asolubility in water of at least 0.001 μg/mL. However, in theseembodiments, no permeation enhancing agent is usually needed tointroduce the compound into the cells.

One can use oligonucleotides containing 1, 2, 3 or more inventionbase(s) to detect a base pair mismatch in a nucleic acid sample usingribonuclease protection assay methods described in U.S. Pat. No.5,589,329. Thus, one can use the invention compounds as described instep (a) or step (b) or step (c) (or, in sequence, steps a, b, or stepsb, c or steps a, b, c) of claim 1 of U.S. Pat. No. 5,589,329 to practicethat claimed method (or to practice necessary steps in the claim 1method, e.g., contacting an RNA probe containing an invention base witha single stranded nucleic acid to form a duplex). One can similarly useoligonucleotides containing an invention base(s) to screen mammaliangenomic DNA samples for insertions, deletions or substitutions usingscreening assay methods described in U.S. Pat. No. 5,589,330. Thus, onecan use the invention compounds as described in step (ii) or step (iii)or step (iv) or step (v) (or, in sequence, steps i, ii, or steps i, ii,iii or steps iii, iv, or steps i, ii, iii, iv, etc.) of claim 1 of5,589,330 to practice that claimed method (or to practice necessarysteps in the claim 1 method, e.g., contacting an oligonucleotidecontaining an invention base with an immobilized genomic DNA sample toform a triplex or duplex). One can similarly use oligonucleotidescontaining an invention base(s) to design a synthesis method for anarray of materials to be synthesized on a substrate using methodsdescribed in U.S. Pat. No. 5,593,839. Thus, one can use the inventioncompounds as described in the first part of the second step or thesecond part of the second step or the third part of the second step orthe fourth part of the second step or (or, in sequence, in the firststep and in the first part of the second step, etc.) of claim 1 of5,593,839 to practice that claimed method (or to practice necessarysteps in the claim 1 method).

One can also use the invention monomer compositions containing a 5'a-³⁵S-thiotriphosphate group or a 5' triphosphate group linked to2',3'-dideoxyribose to perform dideoxy DNA sequencing methods. One mayuse invention monomers in kits that optionally contain buffers orenzymes suitable for DNA sequencing. The invention monomers may beadvantageously used in enzymatic DNA sequencing protocols because theinvention monomers, which act as cytosine surrogates, have a highaffinity for guanosine and may perform better than cytidine 5'triphosphate in sequencing reactions, particularly where the DNA to besequenced contains a high proportion of guanosine residues, which cancause sequencing problems.

Oligonucleotide analogs containing 1, 2, 3 or more invention bases arealso suitable for binding to open complexes in cells or cell lysates ofeukaryotic or prokaryotic cells. Open complexes may also arise insystems comprising at least partially purified cell components, e.g.,RNA polymerase, nucleotide triphosphates, suitable transcriptioncofactors, DNA binding proteins and duplex DNA capable of transcription.Open complexes are regions of single stranded DNA or RNA that occur atleast transiently during duplex nucleic acid metabolism, e.g., duringDNA replication or RNA transcription in the nucleus, cytoplasm, plastidsor mitochondria. Such DNA replication or RNA transcription may involvemetabolism of cellular, viral or other nucleic acids. One can usebinding of invention oligonucleotides to single stranded open complexsequences to affect nucleic acid metabolism, e.g., one may inhibit RNAtranscription or one may use the oligonucleotides, which are optionallylabeled, to detect the presence of open complexes. Such oligonucleotideswill typically comprise about 8 to 30 monomers, usually about 12-21monomers, having a sequence complementary to a single stranded opencomplex region(s) involved in the initiation of a nucleic acid metabolicevent, e.g., initiation of RNA transcription in a promoter ortranscription initiation region. Workers have described open complexesand their formation in vitro and in cells, e.g., Burns, "Biochem. J."317:305-11 1996, Smith, "Proc. Natl. Acad. Sci. USA" 93:8868-72 1996,Jiang, "J Biol Chem" 268:6535-40 1993.

Workers have described other applications that one can practice in asimilar straightforward manner using optionally labeled oligonucleotidescontaining 1, 2, 3 or more invention bases, see e.g., U.S. Pat. Nos.4,910,300, 5,093,232, 5,124,246, 5,202,231, 5,258,506, 5,202,231,5,525,464, 5,578,717, 5,578,715, 5,578,467, 5,591,584, 5,599,932,5,599,668, 5,593,841, 5,578,458, 5,589,332, 5,589,333, 5,589,339,5,589,342, 5,593,830, 5,593,831, 5,593,832, 5,593,836, 5,593,840,5,593,841, 5,593,863, 5,604,097, 5,604,099, 5,605,793, 5,605,794,5,605,796, 5,605,798, 5,605,824, 5,606,047, 5,608,063, 5,614,617,5,594,117, 5,633,364, 5,639,612, 5,639,611, 5,639,608, 5,639,647,5,639,736, 5,639,626, 5,641,631, International Publication Nos. WO97/07246, WO 97/06252, WO 97/06183, WO 97/04787, WO 97/05280, WO96/41017, WO 96/41012, WO 96/40994, WO 96/40996, WO 96/40991, WO96/40992, WO 96/41016, WO 97/04126, WO 97/04129, WO 96/06950 andEuropean Publication No. 761 822. For each of these applications, onewould use an invention oligonucleotide in place of one or more of thedescribed oligonucleotides. To practice these and other typicalapplications, one will typically use an invention oligonucleotide in oneor more of the steps needed to practice the methods described in thesepublications. In many of these applications, one will use an inventionoligonucleotide containing (i) about 7-50, usually about 8-30 linkedmonomers, usually where the oligonucleotide has a uniform polarity, (ii)1, 2 or 3 invention bases, (iii) purine and pyrimidine bases having abase sequence complementary or substantially complementary to a targetsequence, i.e., a defined base sequence having no more than about 1, 2,3 or, for relatively long oligonucleotides (about 35-50-mers), 4 basemismatches, and, optionally, (iv) other moieties or features, which arereadily apparent to the skilled artisan who has read this disclosure,that facilitate using the oligonucleotide in the intended application,e.g., (i) a free hydroxyl group at the 3' terminus for applicationswhere the one wishes to use the invention oligonucleotide as a primer inenzyme-mediated chain elongation applications, (ii) a fluorescent label,enzyme label or radiolabel to facilitate oligonucleotide detection in aparticular assay, (iii) biotin linked to a convenient location such asthe 5' or 3' terminus, or (iv) a free 5' hydroxyl group for enzymaticphosphorylation.

When one adapts the presently claimed compounds to uses known in theart, one will use known hybridization conditions, enzyme (such aspolymerase, RNase or DNase) reaction conditions or detectiontechnologies to design the invention oligonucleotide in a way that doesnot interfere with the intended use, or in a way that improves theintended use. For example, when a previously described assay ordiagnostic method calls for conducting an oligonucleotide hybridizationor enzyme amplification protocol at a specified temperature, one willhave the option of increasing the hybridization or enzyme amplificationtemperature due to the presence of an invention base(s) in theoligonucleotide. Thus, one can use the enhanced binding affinity orenhanced binding specificity property of the invention oligonucleotidesto increase hybridization stringency. Similarly, when one intends to usean invention oligonucleotide in a polymerase chain reaction (PCR)amplification method, one would initially test to see if the presence ofan invention base at the 3' terminus improves the oligonucleotide'sprimer function and then adjust the reaction conditions accordingly,e.g., by altering the primer to target sequence ratio, primerconcentration or by altering the temperature at which one denatures andamplifies the PCR reaction. If the presence of an invention basesignificantly affects polymerase-mediated primer elongation, then onecould choose to design invention oligonucleotides for this use withoutan invention base(s) at the 3' terminal 1, 2 or 3 monomer positions.Skilled artisans routinely design diagnostic or assay protocols bytesting varying temperature, salt composition and concentration, pH,oligonucleotide concentration, enzyme concentration, enzyme reactionbuffer, net oligonucleotide charge, or oligonucleotide base, sugar orlinkage structure during assay development.

Embodiments include a method comprising preparing a series ofoligonucleotides, each having the same base sequence, which sequencecontains 2 or more cytosine bases, where each member of the seriescontains an invention base of structure (3) in place of one or more ofthe cytosine residues. Such oligonucleotides typically comprise 2 toabout 6 cytosine residues. One prepares oligonucleotides containing aninvention base at each cytosine position and optionally one preparesoligonucleotides containing an invention base at each of two cytosinepositions, at each of three cytosine positions and so forth. One usesthis method to determine which oligonucleotide(s), compared to a controlcontaining no invention base(s), has optimal properties for a desiredapplication, e.g., hybridization affinity for use as a probe or optimalantisense activity for inhibiting target gene expression in a cell.

Structure (5) monomers, when triphosphorylated and containing R¹ riboseor deoxyribose derivatives that are chain terminating (e.g. where the 3'position is not hydroxyl), are useful in methods for fluorescentchain-terminating dideoxynucleotide sequencing in the same generalfashion as ddNTPs having other linker-attached fluorophores.

Since oligonucleotide compounds such as those of structure (2) arecapable of participating in Watson-Crick base pairing they will bind tonucleic acids and therefore are useful in detecting the presence ofnucleic acids. Bases of structure (1) in such oligonucleotides willrecognize guanosine as its complementary base in natural nucleic acids.

Invention oligonucleotides, including many structure (2), (2A), (2B) and(2C) oligonucleotides capable of forming high melting duplexes withcomplementary sequences, are useful in numerous applications, includingantisense or codeblocking utilities in vivo or in vitro as well asdiagnostics and probe uses. High melting duplexes are those havingmelting temperatures substantially above the melting temperatures ofoligonucleotide or nucleic acid duplexes of the same sequence thatcontain the ordinary, naturally occurring bases, e.g., adenosine,cytidine, uridine, guanosine, thymidine and the like. "Substantiallyabove" means that the derivative oligonucleotide, when hybridized withits complementary sequence, will not dissociate from the duplex untilthe temperature is raised from about 2 to 40° C., ordinarily about 8 to40° C., above the dissociation temperature of the same oligonucleotidehaving the analogous normal A, C, U, G or T bases, but to no greatertemperature than about 95° C. This is known as the D Tm. Ordinarily, DTm is measured by comparing control oligonucleotide binding tocomplementary RNA or DNA with the binding of test oligonucleotide to thesame RNA or DNA, following, e.g., the method described in Jones et al.,"JOC" 58:2983 (1993).

Some of the invention riboside and deoxyriboside compounds arefluorescent. The compounds remain fluorescent upon incorporation intooligonucleotides and are visible intracellularly, including when boundto target sequences after direct injection or after transfection intocells in accord with known methods.

One can optionally prepare oligonucleotides having tandem arrangementsof the novel bases. In general, such tandem arrangements will containfrom 2 to about 10 invention polycyclic bases, usually 2, 3 or 4, whichcan be the same or different polycycles but generally are the sameinvention polycycle. They also optionally are copolymerized with purineor pyrimidine bases containing known alkynyl substitutions (e.g., U.S.Pat. Nos. 5,645,985 and 5,594,121), in particular pyrimidine basessubstituted at the 5 position with a carbon atom which is bonded toanother atom by a Pi bond, or the fluorescent cytosine derivatives ofInoue et al. (op cit).

The compounds of this invention, or other oligonucleotides capable offorming high melting duplexes (e.g. the Pi bonded bases discussedabove), are useful in improved methods for polymerase chain reaction("PCR") or ligase chain reaction ("LCR") amplification and detection ofnucleic acids. In one embodiment, the high melting oligonucleotides areused as one or both primers in classical PCR or as probes in LCR.Particularly in PCR processes, the elevated melting temperature ofduplexes with high melting primers avoids the need to thermally cyclethe reaction because at these elevated temperatures (about 68 to 95° C.,preferably greater than about 75° C.; the derivative primer willcontinue in at least some proportion to anneal to the target butextension product will not. Ordinary primers will not hybridize and thepolymerase will not initiate transcription until the reaction mixture iscooled to a level at which the primer will anneal to the target sequence(usually, about 55° C.). The elevated temperature that is chosen for usewith the high-melting derivative oligonucleotides (a temperaturesuitable for all of annealing, extension and melting) is one at which asubstantial proportion of the extended primer population (about 10 to 90mole %) is found dissociated from the target, but sufficient unextendedprimer is bound to permit extension. Optimally, this is about from 85 to95° C., ordinarily 92 to 95° C. Alternatively, the optimal temperatureis determined empirically by simply selecting a range of temperatureswithin the melting range of the extended sequence, but within theannealing range of the derivative primers, and measuring the amount ofamplification product to achieve satisfactory levels for the diagnosticor preparative processes at hand. Amplification methods have beendescribed, e.g., U.S. Pat. No. 5,667,974.

An exemplary method to use an invention oligonucleotide to amplify anucleic acid base sequence comprises, providing an inventionoligonucleotide and target nucleic acid sequence that forms a complexhaving a Tm of about 85 to 95° C., optionally heating the complex toabout 85 to 95° C. (e.g., to a temperature within about 5° C. of the Tm)to provide a heated complex, and optionally mixing the heated complexwith a DNA polymerase such as Taq polymerase or other suitable heatstable enzyme. In this method, the complex and the heated complex istypically a duplex. The polymerase reaction will contain cofactors andbuffer conditions suitable for amplification purposes.

It will be understood that the optimal temperature will varyconsiderably depending upon the derivative bases chosen, whether theyare adjacent or separated by other bases, the number of bases in theprimers (the highest annealing temperatures are found with primershaving greater than about 18 bases or base analogs), the proportions ofpyrimidines and purines and the like. The heat stable polymerase usefulin this system is for example Taq polymerase or other suitable heatstable enzyme. Thus, whatever the optimum temperature chosen, theamplification and priming reactions are conducted conventionally but ata substantially constant temperature.

Not only do the oligonucleotides of this invention facilitate PCR or LCRprocesses, the fluorescent properties of the primers also facilitatedetection of the extension products. The extension products are readilyseparated from the unextended primers, e.g. on the basis of molecularweight, and detected by their fluorescence, thereby avoiding stainingwith agents such as ethidium bromide. In some embodiments, thefluorescence is enhanced by using NTP's comprising the fluorescentsubstructures of this invention in primer extension so that thefluorescent NTPs are incorporated into the extension products as well.The polycyclic substructure used in the NTP's may be the same ordifferent than the one incorporated into the primers.

We incorporate herein all citations by reference with specificity.

The following examples further illustrate and do not limit theinvention.

EXAMPLE 1

The following example shows synthesis of representative startingmaterials and intermediates for making invention compounds. ##STR31##Conditions: a: H₂, 10% Pd/C, CH₃ OH, RT; b: HC(OCH₃)₃, methanesulfonicacid, 47.8%; c: Tf₂ NPh, CH₂ Cl₂ /DMF, K₂ CO₃, RT, quantitative; d:HC.tbd.CCH₂ NHTFA, Pd(Ph₃ P)₄, CuI, TEA, DMF, 60%; e: H₂, 10% Pd/C, CH₃COOEt; f: con. NH₄ OH/Dioxane (1/1), RT; g: PhCH₂ OC(O)Cl, pyridine; h:3N HCl/EtOH (1/1), RT, 1 hr or 40° C., 30 min. ##STR32##9-(3'-Aminopropyl)phenoxazine

2-Aminoresorcinol (#1019-43): A ethanol solution (500 mL) of2-nitroresorinol (20 g; 0.129 mmole) was hydrogenated (H₂ in balloon) inthe presence of 10% Pd/C (1 g) at room temperature overnight. Thecatalyst was filtered off through a celite pad. The filtrate wasconcentrated and purified by flash column chromatography, affording15.5g, 96% of 2-aminoresorcinol. ¹ H NMR (DMSO-d₆): δ 8.80 (bs, 2H),6.15-6.30 (m, 3H), 3.85 (bs, 2H).

Compound #1019-45: A trimethyl orthoformate solution (100 mL) of2-aminoresorcinol (6.0 g; 48 mmole) was treated with methanesulfonicacid (4.6 g; 48 mmole). After 30 min stirring at room temperature, thereaction was cooled to 0° C. and was quenched with TEA (4.8 g; 48mmole). The reaction mixture was concentrated and purified by columnchromatography on silica gel, yielding 3.10 g of product, 47.8%. ¹ H NMR(DMSO-d₆): δ 10.3 (bs, 1H), 8.53 (s, 1H), 7.18 (dd, 1H, J=8.0 Hz, J=7.8Hz), 7.12 (d, 1H, J=8.0 Hz), 6.73 (d, 1H, J=7.8 Hz).

Compound #1019-46: A CH₂ Cl₂ /DMF (N,N-dimethylformamide) solution (20mL/10 mL) of #1019-45 (2.90 g; 21.4 mmole) was stirred with solid K₂ CO₃(14.8 g; 107 mmole) at room temperature for 30 min, followed by additionof N-phenyltrifluoromethanesulfonimide (8.50 g; 23.6 mmole). Theresulting mixture was stirred at room temperature overnight, thendiluted with CH₂ Cl₂, washed with water twice, dried, concentrated, andpurified, affording 5.76 g, quantitatively, of # 1019-46. ¹ H NMR(CDCl₃): δ 8.19 (s, 1H), 7.66 (d, 1H, J=8.2 Hz), 7.48 (t, 1H, J=8.2 Hz),7.33 (d, 1H, J=8.2 Hz).

N-TFA-propargylamine (#1019-44): Propargylamine (25 g; 0.45 mmole) wasdissolved in CH₃ OH (500 mL), followed by addition of ethyltrifluoroacetate (84 g; 0.59 mole). The resulting mixture was stirred atroom temperature overnight, then concentrated to dryness. The residuewas redissolved in CH₂ Cl₂ (200 mL), washed with saturated NaHCO₃aqueous solution. The organic phase was isolated, dried, concentrated toa brown residue (liquid). The product #1019-44, 54.0 g was distilledoff, 78.8%. ¹ H NMR (CDCl₃): δ 6.60 (bs, 1H), 4.18 (m, 2H), 2.38 (s,1H).

Compound #1019-97: A DMF solution (12 mL) of compound #1019-46 (4.9 g;18.3 mmole), N-TFA-propargylamine (5.5 g; 36.7 mmole), Pd(PPh₃)₄ (4.3 g;3.7 mmole), CuI (1.78 g; 9.3 mmole) and TEA (3.7 g; 36.7 mmole) wasstirred at room temperature for 24 hours. The reaction mixture wasdiluted with CH₂ Cl₂ (100 mL), stirred with Dowex 1×800 (HCO₃ -- form).The organic phase was washed with water, dried, and purified, yielding2.94 g, 60%, of product #1019-97. ¹ H NMR (CDCl₃): δ 8.18 (s, 1H), 7.62(d, 1H, J=9 Hz), 7.48 (d, 1H, J=9 Hz), 7.36 (t, 1H, J=9Hz), 6.85 (bs,1H), 4.51 (d, 2H, J=3 Hz).

Compound #1019-102: A ethylacetate solution (60 mL) of #1019-97 (1.7 g;6.3 mmole) was hydrogenated in the presence of 10% Pd/C (200 mg) at roomtemperature. The catalyst was filtered off. The filtrate wasconcentrated to dryness, used for next reaction without furtherpurification. ¹ H NMR (CDCl₃): δ 8.25 (bs, 1H), 8.12 (s, 1H), 7.49 (d,1H, J=8.2 Hz), 7.36 (dd, 1H, J=7.6 & 8.1 Hz), 7.20 (d, 1H, J=7.5 Hz),3.25 (q, 2H), 3.07 (t, 2H, J=6.6 Hz), 2.01 (p, 2H).

Compound #1090-05A: A 1.4-dioxane solution (10 mL) of compound #1019-102(1.0 g; 3.6 mmole) was treated with concentrated NHOH (15 mL) at roomtemperature for 16 hrs. The reaction mixture was concentrated todryness. The residue was redissolved in CH₂ Cl₂ (30 mL), containing TEA(0.74 g; 7.35 mmole), cooled to 0° C., followed by addition of benzylchloroformate (0.75 g, 4.4 mmole). The resulting solution was stirred atroom temperature for 4 hrs, washed with H₂ O, dried and purified byflash column chromatography to give 0.81 g, 71% of #1090-05A.

Compound #1090-05: A ethanol solution (10 mL) of compound #1090-05A(0.81 g, 2.6 mmole) was treated with 3 NHCl aqueous solution (10 mL) at40° C. for 1 hr. The reaction mixture was concentrated to dryness,azeotroped with CH₃ CN three times. The crude product was used for nextreaction, without further purification. ¹ H NMR (CDCl₃): δ 7.25-7.40 (m,5H), 7.20 (t, 1H), 6.78-6.90 (m, 2H), 5.07 (s, 2H), 3.17 (t, 2H, J=6.8Hz), 2.67 (t, 2H, J=7.6 Hz), 1.79 (p, 2H).

Compound #1090-18: Compound #1090-15 has been described (Lin, "JACS"117:3873-3874, 1995). A mixture of Compound #1090-15 (1.5 g; 2.6 mmole),#1090-05 (2.6 mmole) and DBU (0.8 g; 5.2 mmole) in CH₂ Cl₂ (30 mL) wasstirred at room temperature overnight. The reaction mixture was washedwith 10% citric acid aqueous solution, dried and purified on silica gelcolumn chromatography, affording 1.21 g, 69% of product. ¹ H NMR(CDCl₃): δ 8.86 (s, 1H), 7.99 (s, 1H), 7.29-7.40 (m, 5H), 7.15 (t, 1H),6.98 (d, 1H, J=8 Hz), 7.78 (d, 1H, J=7 Hz), 6.27 (t, 1H), 5.20-5.23 (m,1H), 5.09 (s, 2H), 4.78 (bs, 1H), 4.30-4.52 (m, 3H), 3.20-3.30 (q, 2H),2.65-2.80 (t+m, 3H), 2.10-2.20 (2 s+m, 7H), 1.80 (p, 2H).

Compound #1090-22: Compound #1019-18 (1.20 g; 1.78 mmole) was treatedwith saturated NH₃ in CH₃ OH (200 mL) at room temperature for 5 days.The reaction mixture was concentrated to dryness and purified by flashcolumn chromatography affording 0.63 g of product #1090-22, 70%. ¹ H NMR(CDCl₃ +10% CD₃ OD): δ 7.30-7.38 (m, 5H), 6.81 (t, 1H, J=7.3 Hz), 6.73(d, 1H, J=7.9 Hz), 6.60 (d, 1H), 6.20 (t, 1H), 5.10 (s, 2H), 4.36-4.40(m, 1H), 3.94-4.0 (m, 1H), 3.70-3.90 (m, 2H), 3.23 (t, 2H), 2.58 (t,2H), 2.30-2.40 (m, 1H), 2.10-2.24 (m, 1H), 1.70-1.82 (m, 2H).

Compound #1090-25: Compound #1090-22 (0.6 g) was dissolved in ethanol(10 mL) and was hydrogenated (H₂ in a balloon) in the presence of 10%Pd/C (50 mg) at room temperature for 4 hrs. The catalyst was filteredoff, washed with CH₃ OH. The filtrate was concentrated and dried toafford 0.3 g, 68% of product. ¹ H NMR (CD₃ OD): δ 7.38 (s, 1H), 6.7-6.82(m, 2H), 6.57 (d, 1H, J=6.3 Hz), 6.23 (dd, 1H, J=6.5 Hz, J=6.7 Hz),4.35-4.38 (m, 1H), 3.80-3.90 (m, 1H), 3.70-3.80 (m, 1H), 2.60-2.80 (m,4H), 2.10-2.30 (m, 2H), 1.75-1.86 (m, 2H).

Compound #1090-26: Compound #1090-25 (0.13 g, 0.34 mmole) was dissolvedin DMF/CH₂ Cl₂ (1 mL/3 mL), followed by addition of9-fluorenylmethyl-N-succinimidylcarbonate (FMOC-NHS, 0.14 g; 0.41mmole). After 1 hr of stirring at room temperature, to the reactionmixture pyridine (140 mg, 1.7 mmole), and DMT-Cl (4,4'-dimethoxytritylchloride; 0.14 g; 0.41 mmole) were added. The resulting mixture wasstirred at room temperature for 2 hrs and then diluted with CH₂ Cl₂,washed with water twice. The organic phase was isolated, dried andpurified by flash column chromatography on silica gel, to give 153 mg,49%, FAB HRMS (high resolution mass spectroscopy) calculated for M+H⁺899.416, found 899.366.

Compound #1090-31: Compound #1090-26 (150 mg; 0.167 mmole) in CH₂ Cl₂ (2mL) was added to a 0° C. cold CH₂ Cl₂ solution (1 mL) of PA (0.25 mL of1M CH₂ Cl₂ solution, 0.25 mmole) and pyridine (66 mg, 0.83 mmole). Theresulting mixture was then gradually warmed to room temperature. After30 minutes, the reaction mixture was diluted with CH₂ Cl₂, washed with 1M TEAB aqueous solution, dried and purified on silica gel, eluted with5% CH₃ OH/CH₂ Cl₂, then 15%, H₂ O in CH₃ CN. The combined fractions ofproduct were concentrated and then partitioned between CH₂ Cl₂ and 1MTEAB aqueous solution dried, yielding 180 mg of H-phosphonatederivative, quantitatively. ##STR33##

Compound #1090-68: A CH₂ Cl₂ --CCl₄ (40 mL/40 mL) solution of5-bromo-3'-5'-diacetyl-2'-deoxyuridine (2.0 g, 5.1 mmole) and triphenylphosphine (2.0 g, 7.6 mmole) was heated at reflux for 3 hrs. Thereaction mixture was cooled to room temperature, followed by addition of2-amino-3-nitrophenol (0.79 g, 5.1 mmole) and DBU (1.6 g, 10 mmole).After stirring at room temperature overnight, the reaction mixture waswashed with 10% citric acid aqueous solution, dried, concentrated andpurified on silica gel. The isolated product contained Ph₃ P═O, wastreated with saturated NH₃ in CH₃ OH for 5 days at room temperature.After removal of all solvent, the residue (crude #1090-51) wasredissolved in CH₃ OH (100 mL), and hydrogenated with H₂ in a balloon inthe presence of 10% Pd/C and 4 N HCl in dioxane (200 mL, 0.8 mmole).After 5 hrs, the catalyst was filtered off, washed with CH₃ OH. Thefiltrate was concentrated and purified by flash column chromatography toafford 0.83 g of #1090-68, in 50% yield for 3 steps. ¹ H NMR (DMSO-Cl₆):δ 9.6 (b, 1H), 6.56 (t, 1H, J=8.0 Hz), 6.22 (d, 1H, J=7.8 Hz), 6.10 (t,1H, J=6.8 Hz), 5.96 (d, 1H, J=7.1 Hz), 5.17 (d, 1H), 4.92-5.08 (m, 3H),4.16-4.20 (m, 1H), 3.70-3.80 (m, 1H), 3.50-3.60 (m, 2H), 2.0 (m, 2H).

Phthalimidoacetaldehyde #1090-70A: A mixture ofN-(2-hydroxyethyl)phthalimide (90 mg; 0.47 mmole), DCC (0.15 g, 0.7mmole), DMSO (1 mL) and dichloroacetic acid (20 mL) was stirred at roomtemperature for 2 hrs. The reaction mixture was diluted with CH₂ Cl₂,washed with H₂ O twice, dried and concentrated. The crude product#1090-70A contained DCC-urea by-product, was used for the next reactionwithout further purification.

Compound #1090-70: A DMF/CH₃ OH (0.5 mL/2 mL) solution of #1090-68 (0.2g, 0.42 mmole), phthalimidoacetaldehyde #1090-70A (0.47 mmole) in CH₃ OH(2 mL) and acetic acid (85 mg, 1.4 mmole) was reacted with sodiumcyanoborohydride (87 mg, 1.4 mmole) at room temperature for 4 hrs. Thereaction mixture was concentrated, and purified on silica gel, yielding243 mg, 78%, yield of compound #1154-70. ¹ H NMR (CDCl₃ +10% DMSO-d₆): δ9.45 (s, 1H), 7.60-7.68 (m, 2H), 7.50-7.58 (m, 2H), 6.51 (t, 1H, J=8.2Hz), 6.12 (d, 1H, J=8.2 Hz), 6.06 (t, 1H, J=6.7 Hz), 5.78 (d, 1H,J=8.0Hz), 4.94 (t, 1H), 4.49-4.52 (m, 1H), 4.18-4.25 (m, 2H), 3.50-3.72(m, 4H), 3.19-3.30 (m, 2H), 1.85-2.05 (m, 2H).

Compound #1090-89: Compound #1090-70 (35 mg; 69 mmole) was dissolved inCH₂ Cl₂ (2 mL) and pyridine (0.5 mL), then reacted with DMT-Cl (28 mg,83 mmole) at room temperature for 3 hrs. The reaction mixture was workedup and purified by flash column chromatography, yielding 44 mg, 78.6% ofcorresponding 5'-O-DMT-derivatives. ¹ H NMR (CDCl₃): δ 7.80-7.90 (m,2H), 7.68-7.75 (m, 2H), 7.18-7.50 (m, 10H), 7.0 (b, 1H), 6.80-6.90 (m,4H), 6.77 (t, 1H), 6.30-6.42 (m, 2H), 5.94 (d, 1H, J=8.0 Hz), 5.50 (bs,1H), 4.50-4.60 (m, 1H), 4.10-4.18 (m, 1H), 3.86-4.0 (m, 2H), 3.25-3.30(2s, 6H), 3.28-3.52 (m, 4H), 2.58-2.68 (m, 1H), 2.20-2.32 (m, 1H).

Compound #1090-91: Compound #1090-89 (44 mg) was converted into3'-H-phosphonate in normal fashion, 52 mg, in 77% yield. ##STR34##

N-CBZ-3-aminopropanol (Compound #1154-74, CBZ; benzyloxycarbonyl): A CH₂Cl₂ solution (300 mL) of 3-aminopropanol (10 g, 0.133 mole) and TEA (20g, 0.2 mole) was cooled to 0° C., followed by slow addition of CH₂ Cl₂solution (25 mL) of benzyl chloroformate (25 g, 0.147 mole). Theresulting solution was gradually warmed to room temperature. Thestirring was continued at room temperature overnight. The reactionmixture was washed with H₂ O, dried and purified by flash columnchromatography (on silica gel, CH₃ OH--CH₂ Cl₂) to yield 16.4 g, 58.9%of title compound. ¹ H NMR (CDCl₃): δ 7.26-7.40 (m, 5H), 5.10 & 5.0-5.10(s+m, 3H), 3.68 (q, 2H), 3.36 (q, 2H), 2.58 (t, 1H), 1.70 (p, 2H).

N-CBZ-2-aminoethanol (Compound #1154-104): To 0° C. cold of CH₂ Cl₂solution (300 mL) of 2-aminoethanol (10.8 g, 0.177 mole) and TEA (26.8g; 0.265 mole) was added slowly a CH₂ Cl₂ solution (50 mL) of benzylchloroformate (33.2 g, 0.194 mole). After complete addition, theresulting solution was gradually warmed to room temperature and stirringwas continued at room temperature overnight. The reaction was worked upand purified by flash column chromatography on silica gel, affording22.8 g in 66% yield of title compound. ¹ H NMR (CDCl₃): δ 7.34-7.40 (m,5H), 5.20-5.32 (m, 1H), 5.14 (s, 2H), 3.74 (q, 2H), 3.37 (q, 2H), 2.38(bs, 1H).

3'-5' -Diacetyl-N⁴ -(2",6"-dihydroxyphenyl)-2'-deoxy-5-bromo-cytidine(Compound #1154-093): A CCl₄ --CH₂ Cl₂ solution (150 mL--150 mL) of5-bromo-3'-5'-diacetyl-2'-deoxyuridine (15 g, 38.3 mmole) and Ph₃ P (15g, 57.5 mmole) was heated at reflux under N₂ for 3 hrs. The reactionmixture was cooled to room temperature, followed by addition of2-amino-resorcinol (5.2 g, 42 mmole) and DBU (8.7 g, 57.5 mmole). Theresulting solution was stirred at room temperature overnight. Thereaction mixture was concentrated to about 1/2 volume, then poured intocitric acid aqueous solution (7.5 g in 300 mL H₂ O) with vigorouslystirring. The precipitate was filtered off, washed with H₂ O, CH₂ Cl₂then CH₃ CN, dried in a vacuum oven overnight, weighed 12.9 g, 67.8%yield of title compound. ¹ H NMR (DMSO-d₆): δ 9.63 (s, 2H), 8.21 (s,1H), 8.0 (s, 1H), 6.89 (t, 1H, J=8.1 Hz), 6.33 (d, 2H, J=8.1 Hz), 6.10(t, 1H, J=7.4 Hz), 5.10-5.17 (m, 1H), 4.12-4.30 (m, 3H), 2.30-2.40 (m,2H), 2.06 &2.03 (2s, 6H).

General procedure for synthesis of compounds 11a-11c and 12a-12c:Compounds 11a-11c: To a CH₂ Cl₂ solution of proper protected aminoalcohol, Ph₃ P (1.5 eq) and diethyl azodicarboxylate (DEAD, 1.5 eq), wasadded compound #1154-093 (1 eq). The resulting mixture was stirred atroom temperature overnight. The reaction mixture was washed with H₂ O,dried and purified by a silica gel (eluted with CH₃ OH--CH₂ Cl₂) toyield compound 11a-11c, normally they contaminated with Ph₃ P═O.However, the crude compound 11a-11c was directly used for compounds12a-12c without further purification.

Compound 12a-12c: Crude compound 11a-11c was treated with saturated NH₃in CH₃ OH at room temperature for 1-3 days. After removal of solvent,the reaction mixture was purified by flash column chromatography onsilica gel (eluted with CH₃ OH--CH₂ Cl₂), to yield compounds 12a-12c.

Compound 12a (1154-106, 1154-111): Compound 12a (2.5 g, 40.8%, 2 steps)was prepared from 1154-093 (6.0 g, 12 mmole) and 1154-104 (3.0 g, 15.3mole). ¹ H NMR (DMSO-d₆): δ 9.82 (bs, 1H), 7.81 (bs, 1H), 7.69 (bs, 1H),7.25-7.36 (m, 5H), 6.82 (t, 1H, J=8.2 Hz), 6.60-6.80 (m, 2H), 6.46 (d,1H, J=8.2 Hz), 6.14 (t, 1H, J=6.7 Hz), 5.22 (d, 1H), 5.06-5.13 (s+s,3H), 4.20-4.25 (m, 1H), 3.94-4.0 (m, 2H), 3.79-3.81 (m, 1H), 3.58-3.65(m, 2H), 3.40-3.50 (m, 2H), 1.98-2.20 (m, 2H).

Compound 12b: Compound 12b (0.30 g; 57.6%) was prepared from 1154-93(0.5 g; 1.0 mmole) and compound 1154-074 (0.31 g; 1.5 mmole). ¹ H NMR(CD₃ OD): δ 7.65 (s, 1H), 7.24-7.36 (m, 5H), 6.79 (t, 1H, J=8.6 Hz),6.54 (d, 1H, J=8.2 Hz), 6.36 (d, 1H, J=8.2 Hz), 6.23 (t, 1H), 5.06 (s,2H), 4.38-4.45 (m, 1H), 3.92-4.11 (m, 3H), 3.81 (q, 2H), 3.38 (t, 2H),2.30-2.40 (m, 1H), 2.10-2.22 (m, 1H).

Compound 11c: Compound 11c (86 mg; 37.7%) was prepared from Compound#1154-093 (200 mg, 0.4 mmole) and N,N-dimethylaminoethanol (43 mg, 0.48mmole). ¹ H NMR (CDCl₃): δ 10.9 (s,1H), 8.49 (s, 1H), 7.97 (s, 1H), 7.11(t, 1H, J=8.2 Hz), 6.77 (d, 1H, J=8.2 Hz), 6.55 (d, 1H, J=8.0 Hz), 6.32(dd, 1H), 5.23-5.27 (m, 1H), 4.40-4.46 (m, 2H), 4.33-4.37 (m, 1H), 4.20(t, 2H, J=5.5 Hz), 2.70-2.80 (m+t, 3H), 2.30 (s, 6H), 2.20 (s, 3H),2.10-2.20 (m+s, 3H).

Compound 13c: Compound 11c (130 mg, 228 mmole) was treated withsaturated NH₃ in CH₃ OH (30 mL). After 2 days at room temperature thereaction mixture was concentrated to dryness to give crude 12c. Thecrude 12c was then dissolved in pyridine (2 mL) containing TEA (89 mg,1.2 mmole), followed by addition of DMT-Cl (115 mg, 340 mmole). After 2hrs at room temperature, the reaction mixture was concentrated,partitioned between CH₂ Cl₂ and saturated NaHCO₃ aqueous solution, driedand purified on silica gel, eluted with 5% CH₃ OH/CH₂ Cl₂, then 10% CH₃OH/CH₂ Cl₂ to yield compound 13c, 60 mg, 34% yield. ¹ H NMR (CDCl₃): δ7.20-7.50 (m, 10H), 6.86 (dd, 4H), 6.75 (t, 1H, J=8.2 Hz), 6.55 (d, 1H,J=8.3 Hz), 6.33 (t, 1H, J=6.1 Hz), 6.28 (d, 1H, J=7.9 Hz), 4.52-4.60 (m,1H), 4.14 (q, 1H), 4.0-4.10 (m, 2H), 3.77 & 3.79 (2s, 6H), 3.32-3.45 (m,2H) 2.62-2.74 (m, 3H), 2.39 (s, 6H), 2.20-2.34 (m, 1H).

Compound 13a (1154-175): A methanol solution (150 mL) of compound 12a(1154-111, 2.2 g, 4.3 mmole) was hydrogenated (H₂ in a balloon) in thepresence of 10% Pd/C at room temperature overnight. The catalyst wasfiltered off, washed with CH₃ OH. The filtrate was concentrated todryness. The crude unprotected 12a derivative was dissolved in DMF/CH₂Cl₂ (10 mL/5 mL), and reacted with FMOC-NHS (1.73 g, 5.1 mmole) at roomtemperature for 1 hr. The reaction mixture was diluted with CH₂ Cl₂ (50mL), containing pyridine (1.0 g, 12.7 mmole), followed by addition ofDMT-Cl (1.8 g, 5.3 mmole). After 2 hrs the reaction mixture was workedup and purified by flash column chromatography, affording 2.87 g, 73% ofcompound 13a. FAB HRMS calculated for C₅₃ H₄₉ N₄ O₁₀ (M+H⁺) 901.345,found 901.344.

Compound 13b: A ethanol solution (15 mL) of compound 12b (1154-077) washydrogenated (H₂ in a balloon) in the presence of 10% Pd/C at roomtemperature for 4 hrs. Catalyst was filtered off. The filtrate wasconcentrated to yield unprotected 12b derivative. Unprotected 12bderivative (125 mg, 0.32 mmole) was dissolved in DMF (2 mL) and reactedwith FMOC-NHS (130 mg, 0.38 mmole) at room temperature for 1 hr. Thereaction mixture was diluted with CH₂ Cl₂ (5 mL) containing pyridine(132 mg, 1.7 mmole), followed by addition of DMT-Cl (135 mg, 0.38mmole). After 2 hrs, the reaction mixture was diluted with CH₂ Cl₂ (10mL), washed with H₂ O, dried and purified by flash column chromatographyto give compound 13b, 86 mg, 29% yield. FAB HRMS calculated for C₅₄ H₅₁N₄ O₁₀ (M+H⁺) 915.360, found 915.361. ##STR35##

Compound #1154-108: 2-Amino-m-cresol (2.1 g, 17.6 mmole) was dissolvedin trimethylorthoformate (15 mL), followed by addition ofmethanesulfonic acid (0.32 g, 3.4 mmole, 20 molar %). The resultingsolution was stirred at room temperature for 3 hrs. The reaction mixturewas neutralized with TEA (0.4 g, 4.1 mmole), concentrated and purifiedon silica gel, eluted with 2% CH₃ OH/CH₂ Cl₂, affording 1.6 g, 72%yield. ¹ H NMR (CDCl₃): δ 8.10 (s, 1H), 7.43 (d, 1H, J=8.2 Hz), 7.31(dd, 1H, J=7.3 Hz, J=8.2 Hz), 7.20 (d, 1H, J=7.3 Hz), 2.68 (s, 3H).

Compound #1154-112: Compound #1154-108 (4.3 g, 32 mmole) was dissolvedin CCl₄ (50 mL), followed by addition of N-bromosuccimide (6.3 g, 35mmole) and AIBN (240 mg). The resulting mixture was refluxed for 2 hrsunder N₂. The reaction mixture was washed with H₂ O, dried, concentratedand purified by flash column chromatography on silica gel, eluted with2% CH₃ OH/CH₂ Cl₂, affording 5.67 g. of product, 82.7%. ¹ H NMR (CDCl₃):δ 8.16 (s, 1H), 7.55 (d, 1H), 7.40-7.50 (m, 2H), 4.90 (s, 2H).

Compound #1154-125: A CH₂ Cl₂ solution (20 mL) of Compound #1154-112(0.28 g, 1.3 mmole), ethylene glycol (0.82 g, 13.2 mmole), silvertriflate (0.5 g, 1.95 mmole) and 2,6-di-t-butyl-4-methylpyridine (0.52g, 2.6 mmole) was stirred at room temperature for 3 hrs. The precipitatewas filtered off, washed with CH₂ Cl₂. The filtrate was washed with H₂O, dried, concentrated and purified on silica gel, eluted with 3% CH₃OH/CH₂ Cl₂, affording 0.17 g, 68% of product #1154-125. ¹ H NMR (CDCl₃):δ 8.14 (s, 1H), 7.55 (d, 1H), 7.36-7.40 (m, 2H), 4.97 (s, 2H), 3.70-3.85(m, 4H),

Compound #1154-126: Compound #1154-125 (170 mg, 0.88 mmole) wasdissolved in CH₂ Cl₂ (5 mL) containing TEA (0.27 g, 2.6 mmole) andtreated with CH₃ SO₂ Cl (0.15 g, 1.32 mmole). After 30 min at roomtemperature, the reaction mixture was washed with H₂ O, dried andconcentrated. The residue was dissolved in DMF (2 mL) followed byaddition of sodium azide (86 mg, 1.3 mmole). The resulting solution washeated at reflux for 2 hrs. The reaction mixture was then partitionedbetween CH₂ Cl₂ and water. The organic phase was isolated, dried andpurified on silica gel, eluted with 1% CH₃ OH/CH₂ Cl2, to afford#1154-126, 162 mg, 84%. ¹ H NMR (CDCl₃): δ 8.10 (s, 1H), 4.99 (s, 2H),3.76 (t, 2H, J=4.8 Hz), 3.45 (t, 2H, J=4.6 Hz).

Compound #1154-132: Compound #1154-126 (1.1 g, 5.0 mmole) was treatedwith 3N HCl aqueous solution (10 mL) and ethanol (10 mL) at 50° C. for 1hr. The reaction mixture was concentrated to dryness, azeotroped withCH₃ CN three times, used for next reaction without further purification.¹ H NMR (DMSO-d₆): δ 10.6 (bs), 6.85-7.20 (m, 3H), 4.60-4.70 (m, 2H),3.60-3.72 (m, 2H), 3.50-3.60 (m, 2H).

Compound #1154-133: 5-Bromo-3',5'-diacetyl-2'-deoxyuridine (0.98 g, 2.5mmole) and Ph₃ P (0.8 g, 3.0 mmole) were dissolved in CCl₄ /CH₂ Cl₂ (10mL/10 mL), and were heated at reflux for 3 hrs. The reaction mixture wascooled to room temperature, followed by addition of #1154-132 (0.62 g,HCl salt, 2.5 mmole) and DBU (0.78 g, 5.1 mmole). The resulting mixturewas then stirred at room temperature for 2 days, washed with 5%, citricacid aqueous solution, dried, concentrated and purified on silica gel.The isolated product contained Ph₃ P═O, without further purification andused for next reaction.

Compound #1154-138: The crude #1154-133 (Theoretically 2.5 mmole) wastreated with saturated NH₃ in CH₃ OH at room temperature for 3 days,concentrated to dryness, and purified in silica gel, yield 0.28 g of 28%yield of product (2 steps). ¹ H NMR (CD₃ OD): δ 7.56 (bs, 1H), 6.78-6.85(m, 2H), 6.64-6.70 (m, 1H), 6.19 (t, 1H, J=6.4 Hz), 4.54 (s, 2H),4.35-4.40 (m, 1H), 3.88-3.94 (m, 1H), 3.60-3.85 (m, 4H), 3.62 (t, 2H),2.22-2.35 (m, 1H), 2.10-2.20 (m 1H).

Compound #1154-148: A CH₃ OH solution (25 mL) of Compound #1154-138 (160mg, 0.38 mmole) was hydrogenated (H₂ in a balloon) in the presence of10% Pd/C at room temperature for 2 hrs. The catalyst was filtered off,washed with CH₃ OH. The filtrate was concentrated to dryness to afford#1154-148. ¹ H NMR (CD₃ OD): δ 7.63 (s, 1H), 6.81-6.86 (m, 2H),6.70-6.74 (m, 1H), 6.22 (t, 1H), 4.65 (s, 2H), 4.35-4.40 (m, 1H),3.89-3.93 (m, 1H), 3.68-3.84 (m, 2H), 3.55 (t, 2H), 2.80-2.90 (m, 2H),2.25-2.36 (m, 1H), 2.10-2.21 (m, 1H).

Compound #1154-149: A DMF solution (2 mL) of compound #1154-148 (170 mg,0.43 mmole) was treated with FMOC-NHS (150 mg, 0.52 mmole). After 2 hrreaction at room temperature the reaction mixture was diluted with CH₂Cl₂ (10 mL) containing pyridine (0.7 g, 8.7 mmole), followed by additionof DMT-Cl (0.22 g, 0.65 mmole). After 1 hr, the reaction was worked upand purified by flash column chromatography, to yield 232 mg, 58.2% of5'-O-DMT-N-FMOC derivative #1154-149. FAB HRMS calculated for C₅₄ H₅₁ N₄O₁₀ (M+H⁺) 915.360, found 915.359. ##STR36##

3'-5'-Diacetyl-O⁴ -sulfonyl-2'-deoxyuridine (#1154-136): A CH₂ Cl₂solution (10 mL) of 3',5'-diacetyl-2'-deoxyuridine (0.76 g, 2.4 mmole),2-mesitylenesulfonyl chloride (1.0 g, 4.8 mmole), TEA (1.23 g, 12.1mmole) and catalytic amount of DMAP (0.1 g) was stirred at roomtemperature overnight. The reaction mixture was diluted with CH₂ Cl₂,washed with 5% citric acid aqueous solution, dried and purified onsilica gel, eluted with CH₂ Cl₂, 35% ethyl acetate in CH₂ Cl₂, to givethe title compound, 0.76 g in 63% yield. ¹ H NMR (CDCl₃): δ 8.0 (d, 1H,J=9 Hz), 6.99 (s, 10 2H), 6.13 (d, 1H, J=9 Hz), 6.07 (t, 1H, J=6.0 Hz),5.15-5.20 (m, 1H), 4.33 (s, 3H), 2.70-2.85 (m+s, 7H), 2.73 (s, 3H), 2.09(s, 3H), 1.98-2.07 (m+s, 4H).

3'-5'-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxyanediyl)-9"-(aminoethoxy)phenoxazine(#1154-135): Compound 14 (see preparation of 12a and 13a) was dissolvedin DMF/pyridine (2 mL/2 mL) followed by addition of1,1,3,3-tetraisopropyl-dichlorodisilane (0.28 g, 0.91 mmole). Thestirring was continued at room temperature for 3 hr. The reactionmixture was concentrated to dryness. The residue was used for nextreaction without further purification.

Compound #1154-137: A CH₂ Cl₂ solution (10 mL) of compound #1154-136(0.3 g, 0.61 mmole), compound #1154-135 (crude, 0.61 mmole), and DBU(0.46 g, 3.0 mmole) was stirred at room temperature overnight. Thereaction mixture was washed with 5% citric acid aqueous solution, dried,and purified by flash column chromatography (silica gel, CH₃ OH/CH₂Cl₂), to give 0.21 g, 38% of product #1154-137. FAB LRMS calculated forC₄₂ H₆₁ N₆ O₁₃ Si₂ (M+H⁺) 912, found 913.

Compound #1154-144: Compound #1154-137 (0.20 g, 0.22 mmole) in THF 1 mL,was treated with 1 M Bu₄ NF in CH₂ Cl₂ solution (0.87 mL, 0.87 mmole) atroom temperature for 30 min, then concentrated to dryness. The residuewas redissolved in CH₂ Cl₂ (5 mL) containing pyridine (173 mg; 2.2mmole), followed by addition of DMT-Cl (330 mg, 1 mmole). After 2 hrs,the reaction mixture was washed with saturated NaHCO₃ aqueous solution,dried, and purified by flash column chromatography (silica gel, CH₃OH/CH₂ Cl₂) to yield 0.14 g, 65% of product #1154-144. FAB HRMScalculated for C₅₁ H₅₃ N₆ O₁₄ (M+H⁺) 973.362, found 973.364.

EXAMPLE 2

Antisense inhibition of T-antigen expression. The tested DNAoligonucleotide analogs, had the base sequence shown in Table I below.This sequence is complementary to a the 12 base RNA sequence expressedby the large SV40 virus large T antigen gene, 5' GTA GTG AGG AGG 3' (SEQID NO. 1). In the tested oligonucleotides, which are shown in Table I,each linkage was a phosphorothioate linkage and all sugars were2'-deoxyribose.

The tested oligonucleotides had the base sequences shown in Table I. Inthe Table I oligonucleotides, bases were designated as follows.

    ______________________________________                                        Base                                                                                             abbreviation base                                          ______________________________________                                        C                 5-methylcytosine                                              U 5-(1-propynyl)uracil                                                        A adenine                                                                     T thymine                                                                     D 5-(1-propynyl)cytosine                                                      Z structure (58)                                                              V structure (61)                                                              X structure (57)                                                              Y structure (60)                                                            ______________________________________                                    

Structures of tricyclic bases described in this example and in followingexamples are as follows. ##STR37##

The T_(m) values obtained from the RNA hybrids and the intracellularIC₅₀ values for T-antigen inhibition derived from microinjectionanalysis are shown below. In all cases, β-galactosidase inhibition wasconcurrently tested as an internal control and we saw no inhibition at 5μM, the highest concentration tested. We measured inhibition of Tantigen expression in CV-1 cells in tissue culture essentially asdescribed (Wagner et al., "Science" 260:1510-1513 1993). We measured theΔT_(m) (° C.) values relative to control ODN1 essentially as described(Jones et al. "JOC" 58:2983 1993).

                                      TABLE I                                     __________________________________________________________________________    ODN                          ΔT.sub.m                                                                    IC.sub.50                                      # BASE ODN sequence 5' to 3' (° C.)* (μM)                         __________________________________________________________________________     1 C control                                                                           CCU-CCU-CAC-UAC                                                                          SEQ ID NO. 2                                                                           (65.0)                                                                            0.5-1.0                                         2 D control DDU-DDU-DAD-UAD SEQ ID NO. 3  +13.0 0.25                          3 (57) XCU-CCU-CAC-UAC SEQ ID NO. 4  +2.5 1.0                                 4 (57) CXU-CCU-CAC-UAC SEQ ID NO. 5  +9.5 0.05                                5 (57) CCU-XCU-CAC-UAC SEQ ID NO. 6  +6.5 0.1                                 6 (57) CCU-CXU-CAC-UAC SEQ ID NO. 7  +9.0 0.05                                7 (57) CCU-CCU-XAC-UAC SEQ ID NO. 8  +6.5 0.05-0.1                            8 (57) CCU-CCU-CAX-UAC SEQ ID NO. 9  +5.0 0.1                                 9 (57) CCU-CCU-CAC-UAX SEQ ID NO. 10 +5.5 2.0                                10 mismatch CCX-CCU-UAC-UAC SEQ ID NO. 11 -12.5 >0.5                          11 T control CCT-CCT-CAC-TAC SEQ ID NO. 12 -11.0 >1.0                         12 T-(57) CCT-CCT-XAC-TAC SEQ ID NO. 13 -2.0 0.25                             16 D CCU-CCU-DAC-UAC SEQ ID NO. 14 0 0.5-1                                    17 (60) CCU-CCU-YAC-UAC SEQ ID NO. 15 +0.5 >0.5                               18 (58) CCU-CCU-ZAC-UAC SEQ ID NO. 16 +5.0 0.3                                19 (61) CCU-CCU-VAC-UAC SEQ ID NO. 17 +1.0 0.35                             __________________________________________________________________________     *ΔT.sub.m Relative to control ODN1                                 

The results above show that the presence of an invention base elicitspotent and specific antisense inhibition of target gene expression(compare ODN1 vs. ODN4 and ODN6). Incorporation of one invention base atan internal C position resulted in a potency enhancement which exceedsthat obtained from the substitution of seven 5-(1-propynyl)cytosinebases for 5-methylcytosine (compare ODN2 and ODN4). The ODN12 resultsshowed that oligonucleotides containing limited base substitutions andonly a single invention base may have significant antisense potency.Phosphorothioate linked oligodeoxynucleotides containingpropynyl-substituted pyrimidine bases were previously the most potentclass of antisense agents that workers had described.

Oligonucleotides containing a base of structure (3) at an internalposition in the oligonucleotide where R² was (a) the (S) isomer of--O--CH₂ --C*H(CH₃)--NH₂ or (b) the (R) isomer of --O--CH₂--C*H(CH₃)--NH₂ were prepared and tested for binding affinity in asimilar manner. Both oligonucleotides had an increased binding affinitycompared to the control oligonucleotide.

EXAMPLE 3

Antisense inhibition of p27^(kip1) expression. The sequence of ODN13 was5' UGGCUCUCCUGCGCC 3' (SEQ ID NO. 18) and it contained phosphorothioatelinkages, all sugars were 2'-deoxyribose, all G positions containedguanine, all U positions contained 5-(1-propynyl)uracil and all Cpositions contained 5-(1-propynyl)-cytosine. ODN14 had the same sequenceand base composition as ODN13, except that all C positions contained5-methylcytosine instead of 5-(1-propynyl)cytosine and thymidine insteadof 5-(1-propynyl)uracil. ODN15 had the same sequence and basecomposition as ODN14, except that the 9th C position from the 5' endcontained a (57) base instead of 5-methylcytosine. Each ODN was testedfor its capacity to inhibit p27 gene expression in CV-1 cells in cellculture using cationic lipid to deliver the oligonucleotides into thecells, essentially as described (Coats "Science" 272:877-880, 1996).

    ______________________________________                                                ODN  IC.sub.50 (nM)                                                   ______________________________________                                                13   10                                                                 14 >20                                                                        15 <5                                                                       ______________________________________                                    

The results showed that ODN15 containing one (57) base is more potentthan the all-propyne derivative ODN13. Previously,phosphorothioate-linked oligonucleotides containing5-1(propynyl)-modified pyrimidine bases were the among the most potentreported class of antisense agents.

ODN13 was tested in rats and, in a 10 day toxicological evaluation, itwas found that the MTD (maximum tolerated dose) was 0.6 mg/kg/d i.v.ODN15 was tested in the same manner with no toxicity observed at a doseof 6.0 mg/kg/d i.v.

EXAMPLE 4

Increased oligonucleotide binding affinity and specificity. We made a10-mer oligonucleotide, ODN22, having the base sequence, 5' TCTCCCTCTC3' (SEQ ID NO. 19). ODN22 contained only phosphorothioate linkages, allsugars were 2'-deoxyribose, bases designated T were thymine, basesdesignated C were 5-methylcytosine. ODN23 was the same as ODN22, exceptthat the 5th base position from the 5' end contained5-(1-propynyl)cytosine (base designated "D" in Table IV). ODN24 was thesame as ODN22, except that the 5th base position from the 5' endcontained a structure (60) base. ODN25 was the same as ODN22, exceptthat the 5th base position from the 5' end contained a structure (55)base. ODN26 was the same as ODN22, except that the 5th base positionfrom the 5' end contained a structure (57) base. ODN27 was the same asODN22, except that the 5th base position from the 5' end contained astructure (59) base. ODN28 was the same as ODN22, except that the 5thbase position from the 5' end contained a structure (58) base. ODN29 wasthe same as ODN22, except that the 5th base position from the 5' endcontained a structure (61) base. ODN30 was the same as ODN22, exceptthat the 5th base position from the 5' end contained a structure (65)base.

We measured the T_(m) (° C.) of ODN22-ODN30 using 4 differentoligonucleotides: A complementary RNA oligonucleotide (ODN31), an RNAoligonucleotide (ODN32) having adenine at the 11th position from its 5'end, i.e., a single A: test base mismatch, a complementary DNAoligonucleotide (ODN33), and a DNA oligonucleotide (ODN34) havingadenine at the 11th position from its 5' end. We measured the ΔT_(m) (°C.) values are relative to control ODN22 essentially as described (Jones"JOC" 58:2983 1993). The results are shown in Table II.

                                      TABLE II                                    __________________________________________________________________________    Thermal Denaturation Data for 9-Modified Phenoxazine ODNs                       Target DNA (33)/RNA (31): 5'-AAA-AAG-AGA-GGG-AGA (SEQ ID NO. 21, 22)         Target DNA (34)/RNA (32): 5'-AAA-AAG-AGA-GAG-AGA (SEQ ID NO. 23, 24)                              ΔTm     ΔTm                                                                      ODN test base 31 ΔTm* 32                                               (31-32) 33 ΔTm* 34 (33-34)           __________________________________________________________________________    22  C (control)                                                                          61.5                                                                             --  42.5                                                                             19.0                                                                              50.5                                                                             --  32.0                                                                             18.5                                         23 D (control) 65.0  3.5 44.5 20.5 54.0  3.5 33.0 21.0                        24 (60) (control) 66.5  5.0 50.0 16.5 57.0  6.5 44.5 12.5                     25 (55) 73.5 12.0 56.0 17.5 63.5 13.0 44.0 19.0                               26 (57) 77.5 16.0 52.0 25.5 68.5 18.5 43.0 25.5                               27 (59) 74.0 12.5 51.0 23.0 -- -- -- --                                       28 (58) 73.5 12.0 52.5 21.0 -- -- -- --                                       29 (61) 70.5  9.0 55.0 15.5 -- -- -- --                                       30 (65) 61.5 0  44.0 17.5 -- -- -- --                                       __________________________________________________________________________     *DTm relative to ODN22.                                                  

This data demonstrates the enhancement in melting point afforded byoligonucleotides containing invention bases. The increased ΔTm(31-32)and ΔTm(33-34) values obtained with invention bases (57), (58) and (59)indicate that these invention bases have an increased bindingspecificity compared to 5-methylcytosine or 5-(1-propynyl)cytosine.

EXAMPLE 5

Increased potency of gene expression inhibition. We made a 20-merphosphorothioate-linked DNA oligonucleotide, 5'TCC-CGC-XTG-TGA-CAT-CGA-TT 3' (SEQ ID NO. 25), where X was a structure(57) base. The oligonucleotide was complementary to the 3' untranslatedregion of the c-raf mRNA. A control oligonucleotide had the samesequence except that the X base was replaced with cytosine. Eacholigonucleotide was tested to determine its potency at inhibitingexpression c-raf gene expression essentially as described (Monia "NatureMed" 2:668-675 1966, WO 97/32604). Briefly, a range of concentrations ofeach oligonucleotide was transfected into A549 small lung carcinomacells on two consecutive days, followed by preparing cell extracts 48hours after the first transfection. Immunoblot assay for c-raf proteinexpression showed the control oligonucleotide reduced c-raf proteinexpression with an IC₅₀ of about 20 nM. The test oligonucleotidecontaining the structure (57) base in place of cytosine was at least20-fold more potent and had an IC₅₀ of less than 1 nM.

Similar assays using an oligonucleotide containing about 8-18 bases thatare complementary to raf or c-raf, e.g., the oligonucleotide sequenceused in this example or a shortened version thereof, is accomplished ina similar manner using invention oligonucleotides containing 1, 2 or 3invention bases having an R² moiety that increases binding affinitycompared to a control oligonucleotide containing cytosine.

We claim:
 1. A compound having the structure (1): ##STR38## andtautomers, solvates and salts thereof, wherein R¹ is an oligonucleotide,a protecting group, a linker or --H;R² is A(Z)_(X1), wherein A is aspacer and Z independently is a label bonding group optionally bonded toa detectable label, but R² is not amine, protected amine, nitro orcyano; R²⁷ is independently --CH═, --N═, --C(C₁ -C₈ alkyl)═ or--C(halogen)═, but no adjacent R²⁷ are both --N═, or two adjacent R²⁷are taken together to form a ring having the structure, ##STR39## whereR^(a) is independently --CH═, --N═, --C(C₁₋₈ alkyl)═ or --C(halogen)═,but no adjacent R^(a) are both --N═; R³⁴ is --O--, --S-- or --N(CH₃)--;and X1 is 1, 2 or
 3. 2. The compound of claim 1 wherein R² is --R^(2C)--R^(2D), wherein R^(2C) is a short spacer chain and R^(2D) is ahydrogen bond donor moiety or a moiety having a net positive charge ofat least about +0.5 at pH 6-8 in aqueous solutions.
 3. The compound ofclaim 1 wherein R² is --R⁶ --(CH₂)_(t) NR⁵ C(NR⁵)(NR³)₂, --R⁶ --CH₂--CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_(v) --N(R³)₂, --R⁶ --(CH₂)_(t) --N(R³)₂,--(CH₂)₁₋₂ --O--(CH₂)_(t) --N(R³)₂, ##STR40## R³ is independently --H,--CH₃, --CH₂ CH₃, --(CH₂)_(w) --N(R³³)₂ or a protecting group, or bothR³ together are a protecting group, or when R² is --R⁶ --(CH₂)_(t)--N(R³)₂, one R³ is --H, --CH₃, --CH₂ CH₃, a protecting group or--(CH₂)_(w) --N(R³³)₂ and the other R³ is --H, --CH₃, --CH₂ CH₃,--(CH₂)_(w) --N(R³³)₂, --CH(N[R³³ ]₂)--N(R³³)₂, ##STR41## R⁵ isindependently H or a protecting group; R⁶ is independently --S--, --NR⁵--, --O-- or --CH₂ --;R⁷ is independently linear alkyl having 1, 2, 3 or4 carbon atoms optionally substituted with one --CH═CH--, --C.tbd.C-- or--CH₂ --O--CH₂ -- moiety, or R⁷ is cyclic alkyl having 3, 4 or 5 carbonatoms, wherein one of the linear alkyl carbon atoms is optionallysubstituted with a single --CH₃, --CN, ═O, --OH or protected hydroxyl,provided that the carbon atoms in any --CH═CH-- or --CH₂ --O--CH₂ --moiety are not substituted with ═O, --OH or protected hydroxyl; R⁸ islinear alkylene having 1 or 2 carbon atoms wherein one alkylene carbonatom is optionally substituted with a single --CH₃, --CN, ═O, --OH orprotected hydroxyl, or R⁸ is absent; R²⁸ is independently --CH₂ --,--CH(CH₃)--, --CH(OCH₃)--, --CH(OR⁵)-- or --O--, but both are not --O--;R²⁹ is independently --N--, --N(CH₃)--, --CH--, --C(CH₃)--, but both arenot --N(CH₃)--; R³⁰ is --H or --N(R³)₂ ; R³¹ is the side chain of anamino acid; R³³ is independently --H, --CH₃, --CH₂ CH₃ or a protectinggroup; R³⁵ is H, C₁ -C₄ alkyl or a protecting group; R³⁶ is --H, --CH₃,--CH₂ CH₃, a protecting group or an optionally protectedmonosaccharide;t is 1, 2, 3 or 4, but when R⁶ is --O--, --S-- or --NR⁵--, t is 2, 3 or 4; v is independently 0, 1 or 2; and w is independently1 or
 2. 4. The compound of claim 3 wherein R² is --CH₂ --(CH₂)_(t)N(R³)₂, --NR⁵ --(CH₂)_(t) N(R³)₂, --S--(CH₂)_(t) N(R³)₂, --O--(CH₂)_(t)N(R³)₂, --O--(CH₂)_(t) NR⁵ C(NR⁵)(NR³)₂, --(CH₂)₁₋₂ --O--(CH₂)_(t)N(R³)₂, --R⁶ --CH₂ --CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_(v) --N(R³)₂, --R⁶--(CH₂)_(t) --NR⁵ C(NR⁵)(NR³)₂, or --CH₂ (CH₂)_(t) NR⁵ C(NR⁵)(NR³)₂. 5.The compound of claim 4 wherein t is
 2. 6. The compound of claim 5wherein R³ independently is --H, --CH₃ --C₂ H₅ or a protecting group. 7.The compound of claim 6 wherein R² is --O--(CH₂)₂ --NH₂, --O--(CH₂)₃--NH₂, --O--(CH₂)₂ --N(CH₃)₂, --O--(CH₂)₃ --N(CH₃)₂, --O--(CH₂)₂--NHCH₃, --O--(CH₂)₃ --NHCH₃, --O--CH₂ --CH(CH₃)--NH₂, --CH₂ --O--(CH₂)₂--NH₂, --CH₂ --O--(CH₂)₃ --NH₂ or --(CH₂)₂ --O--(CH₂)₂ --NH₂.
 8. Thecompound of claim 3 wherein t is 2 or
 3. 9. The compound of claim 1wherein R¹ comprises --H, an optionally protected monosaccharide,hydroxyl, phosphate or hydrogen phosphonate.
 10. The compound of claim 1wherein R¹ is optionally protected 2'-deoxy-R²¹ -substituted ribose,2'-deoxyribose or ribose, wherein R²¹ is H, --OH, halogen or a moietythat enhances the nuclease stability of an oligonucleotide containingthe optionally protected 2'-deoxy-R²¹ -substituted ribose,2'-deoxyribose or ribose.
 11. The compound of claim 1 having thestructure designated by the numbers selected from the group consistingof (104), (105), (133), (134), (111), (112), (113), (115), (135), (136),(137), (138), (139), (120), (121), (121A), (143), (122), (123), (125),or (126): ##STR42## wherein R¹ is an optionally protectedmonosaccharide;R^(2A) is --OH; R⁵ is independently --H or a protectinggroup; R⁶ is --O--, --S--, --NH-- or --CH₂ --, R²¹ is H, --OH, halogenor a moiety that enhances the nuclease stability of an oligonucleotide;R²⁴ is a halogen; R²⁷ is independently --CH═, --N═, --C(C₁ -C₈ alkyl)═or --C(halogen ═, but no adjacent R²⁷ are both --N═, or two adjacent R²⁷are taken together to form a ring having the structure, ##STR43## whereR^(a) is independently --CH═, --N═, --C(C₁₋₈ alkyl)═ or --C(halogen)═,but no adjacent R^(a) are both --N═; R³⁴ is --O--, --S-- or --N(CH₃)--;R³⁷ is --O--, --CH₂ -- or --CF₂ --; R⁴⁷ is --O-- or --S--; R⁵⁰ is --CH₂--, --C(O)--, --(CH₂)₂ --O--(CH₂)₂ --, --(CH₂)₂ --NR⁵ --(CH₂)₂ --,--(CH₂)₂ --S--(CH₂)₂ --, --CH(N(R⁵)₂)--, --CH(COOR⁵)-- or --C(CH₃)--,--C(C₂ C₅)-- but adjacent moieties are not C(O); R⁵² is--(CHR^(52A))--(R^(52B))--CHR^(52A) --, --CHR^(52A) --O--CHR^(52A) --,--CHR^(52A) --S--CHR^(52A) --, --CHR^(52A) --NR⁵ --CHR⁵² A--, C₁ -C₁₀alkylene optionally substituted with 1 or 2 moieties selected from thegroup consisting of C₁ -C₆ alkyl, --OR⁵, ═O, --NO₂, --N₃, --CN, --COOR⁵,or --N(R⁵)₂, wherein any heteroatom is separated from the nitrogen atomsthat R⁵² is linked to by one methylene and one or more --CHR^(52A) --;R^(52A) is --H or C₁ -C₆ alkyl; R^(52B) is a bond; R⁵⁹ is --R⁶ --R⁶⁰ --;R⁶⁰ is --(CH₂)_(Z3) --(R⁶¹)_(Z1) --(CH₂)_(Z2) --; R⁶¹ is --O--, --S--,--NR⁵ --, --C(O)--, --CH₂ --O--CH₂ --, --CH₂ --NR⁵ --CH₂ -- or CH₂--S--CH₂ --; Z1 is 0 or 1; Z2 is 1, 2 or 3; Z3 is 1, 2 or 3; Y is 1, 2,3 or 4; CBZ is carboxybenzoyl; Fmoc is 9-fluorenylmethoxycarbonyl; iPris isopropyl; and Ac is acetyl.
 12. The compound of claim 1 wherein R¹is an oligonucleotide having the structure (2): ##STR44## wherein D is--OH, protected --OH, an oligonucleotide coupling group or a solidsupport;D¹ is an oligonucleotide coupling group, --OH, protected --OH ora solid support, wherein D¹ is bonded to one 2' or 3' position in theoligonucleotide of structure (2) and the adjacent 2' or 3' position instructure (2) is substituted with R²¹, provided that D and D¹ are notboth an oligonucleoide coupling group or they are not both a solidsupport; R⁴ is independently a phosphodiester linkage or aphosphodiester substitute linkage, wherein R⁴ is bonded to one 2' or 3'position in the structure (2) oligonucleotide and the adjacent 2' or 3'position in structure (2) is substituted with R²¹ ; R²¹ is independently--H, --OH, halogen or a moiety that enhances the oligonucleotide againstnuclease cleavage; R³⁷ is independently --O--, --CH₂ --, --CF₂ --; n isan integer from 0 to 98; and B independently is a purine or pyrimidinebase or a protected derivative thereof, provided that at least one B isa base of structure (3) ##STR45##
 13. The compound of claim 12 whereinR⁴ is independently 3'-O--P(S)(S)--O-5', 3'-O--P(S)(O)--O-5',3'-O--P(O)(O)--O-5', 3'-O--P(Me)(O)--O-5', 3'-NH--P(O)(O)--O-5',3'-S--CH₂ --O-5', 2'-S--CH₂ --O-5', 3'-O--CH₂ --O-5', 2'-O--CH₂ --O-5',3'O--P(Me)(S)--O-5', 3'-CH₂ --N(CH₃)--O-5', 2'-CH₂ --N(CH₃)--O-5', or3'-R³⁸ --P(N₂)(O)--O-5', wherein R³⁸ independently is --O--, --CH₂ -- or--NH--;R³⁹ is a protecting group; R⁴⁰ independently is hydrogen, aprotecting group, C₁ -C₁₂ alkyl optionally substituted with one, or two--O--, --C(O)--, --OC(O)--, --C(O)O--, --OR⁴², --SR⁴³, --C(O)NR³⁹ --,--C(O)N(R⁴¹)₂, --NR⁴¹ --, --N(R⁴¹)₂, halo, --CN, or --NO₂ moieties, orboth R⁴⁰ together with the nitrogen atom to which they are attached form##STR46## or both R⁴⁰ together are a protecting group; R⁴¹ independentlyis hydrogen, a protecting group, alkyl (C₁ -C₄ or both R⁴¹ together area protecting group; R⁴² is hydrogen or a protecting group; R⁴³ is C₁₋₆alkyl or a protecting group; and R⁴⁵ is --H, a counter ion or ##STR47##R⁴⁶ is alkyl containing 1-8 carbon atoms.
 14. The compound of claim 12wherein R² is --R⁶ --(CH₂)_(t) NR⁵ C(NR⁵)(NR³)₂, --R⁶ --CH₂ --CHR³¹--N(R³)₂, --R⁶ --(R⁷)_(v) --N(R³)₂, --R⁶ --(CH₂)_(t) --N(R³)₂,--(CH₂)₁₋₂ --O--(CH₂)_(t) --N(R³)₂, ##STR48## R³ is independently --H,--CH₃, --CH₂ CH₃, --(CH₂)_(w) --N(R³³)₂ or a protecting group, or bothR³ together are a protecting group, or when R² is --R⁶ --(CH₂)_(t)--N(R³)₂, one R³ is --H, --CH₂ CH₃, a protecting group or --(CH₂)_(w)--N(R³³)₂ and the other R³ is --H, --CH₃, --CH₂ CH₃, --(CH₂)_(w)--N(R³³)₂, --CH(N[R³³ ]₂)--N(R³³)₂, ##STR49## R⁵ is independently H or aprotecting group; R⁶ is independently --S--, --NR⁵ --, --O-- or --CH₂--;R⁷ is independently linear alkyl having 1, 2, 3 or 4 carbon atomsoptionally substituted with one --CH═CH--, --C.tbd.C-- or --CH₂ --O--CH₂-- moiety, or R⁷ is cyclic alkyl having 3, 4 or 5 carbon atoms, whereinone of the linear alkyl carbon atoms is optionally substituted with asingle --CH₃, --CN, ═O, --OH or protected hydroxyl, provided that thecarbon atoms in any --CH═CH-- or --CH₂ --O--CH₂ -- moiety are notsubstituted with ═O, --OH or protected hydroxyl; R⁸ is linear alkylenehaving 1 or 2 carbon atoms wherein one alkylene carbon atom isoptionally substituted with a single --CH₃, --CN, ═O, --OH or protectedhydroxyl, or R⁸ is absent; R²⁸ is independently --CH₂ --, --CH(CH₃)--,--CH(OCH₃)--, --CH(OR⁵)-- or --O--, but both are not --O--; R²⁹ isindependently --N--, --N(CH₃)--, --CH--, --C(CH₃)--, but both are not--N(CH₃)--; R³⁰ is --H or --N(R³)₂ ; R³¹ is the side chain of an aminoacid; R³³ is independently --H, --CH₃, --CH₂ CH₃ or a protecting group;R³⁵ is H, C₁ -C₄ alkyl or a protecting group; R³⁶ is --H, --CH₃, --CH₂CH₃, a protecting group or an optionally protected monosaccharide;t is1, 2, 3 or 4, but when R⁶ is --O--, --S-- or --NR⁵ --, t is 2, 3 or 4; vis independently 0, 1 or 2; and w is independently 1 or
 2. 15. Thecompound of claim 14 wherein R² is --CH₂ --(CH₂)_(t) N(R³)₂, --NR⁵--(CH₂)_(t) N(R³)₂, --S--(CH₂)_(t) N(R³)₂, --O--(CH₂)_(t) N(R³)₂,--O--(CH₂)_(t) NR⁵ C(NR⁵)(NR³)₂, --(CH₂)₁₋₂ --O--(CH₂)_(t) N(R³)₂, --R⁶--CH₂ --CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_(v) --N(R³)₂, --R⁶ --(CH₂)_(t) --NR⁵C(NR⁵)(NR³)₂, or --CH₂ (CH₂)_(t) NR⁵ C(NR⁵)(NR³)₂.
 16. The compound ofclaim 15 wherein t is 2 or
 3. 17. The compound of claim 16 wherein R³independently is --H, --CH₃, --C₂ H₅ or a protecting group.
 18. Thecompound of claim 17 wherein R² is --O--(CH₂)₂ --NH₂, --O--(CH₂)₃ --NH₂,--O--(CH₂)₂ --N(CH₃)₂, --O--(CH₂)₃ --N(CH₃)₂, --O--(CH₂)₂ --NHCH₃,--O--(CH₂)₃ --NHCH₃, --O--CH₂ --CH(CH₃)--NH₂, --CH₂ --O--(CH₂)₂ --NH₂,--CH₂ --O--(CH₂)₃ --NH₂ or --(CH₂)₂ --O--(CH₂)₂ --NH₂.
 19. The compoundof claim 12 wherein R²¹ is independently --H, --OH, halogen, protectedhydroxyl, --O-methyl, --O-ethyl, --O-n-propyl, --O-allyl, --O--(CH₂)₂OH, --O--(CH₂)₃ OH, --O--(CH₂)₂ F, --O--(CH₂)_(s) R⁶⁵, --O--(CH₂)₂--[O--(CH₂)₂ ]_(r) R⁶⁵, --O--(CH₂)_(r) --O--(CH₂)_(r) --O--(CH₂)_(r)--R⁶⁵), --NH-methyl, --NH-ethyl, --NH-n-propyl, --NH--(CH₂)₂ OH,--NH--(CH₂)₃ OH, --NH--(CH₂)_(s) R⁶⁵, --S-methyl, --S-ethyl,--S-n-propyl, --S-allyl, --S--(CH₂)₂ OH, --S--(CH₂)₃ OH --S--(CH₂)₂ F,--S--(CH₂)_(s) R⁶⁵, or --S--(CH₂)₂ --[O--(CH₂)₂ ]_(r) R⁶⁵, whereinR⁶⁵ is--H, --F, --OH, --OCH₃, --NH₂, --SH, protected hydroxyl, protected aminoor protected thiol;r is 1, 2, 3 or 4; and s is 2, 3, 4, 5, 6, 7 or 8.20. The compound of claim 19 wherein R²¹ is independently --H, --OH,--F, protected hydroxyl, --OCH₃, --O--CH₂ CH₃, --O--CH₂ CH₂ OH, --O--CH₂CH₂ F, --O--CH₂ CH₂ CH₃, --O--CH₂ CH₂ CH₂ OH, --O--CH₂ CH₂ CH₂ F,--O--CH₂ CF₂ H, --O--CH₂ CF₃ or --O--CH₂ CH₂ --O--CH₃.
 21. The compoundof claim 12 wherein B independently are selected from the groupconsisting of a base of structure (3), guanosine, adenine, thymine,uracil, cytosine, 5-methylcytosine, 5-(1-propynyl)uracil,5-(1-propynyl)cytosine, 5-(1-butynyl)uracil therefor5-(1-butynyl)cytosine.
 22. The compound of claim 12 wherein D¹ isH-phosphonate, a methylphosphonamidite, a β-cyanoethylphosphoramidite ora phosphoramidite.
 23. A compound having the structure (4) ##STR50## andtautomers, solvates and salts thereof wherein, R¹, R² and R²⁷ have themeanings given in claim 1;R²⁴ is a halogen; R²⁵ is --SH, --OH, ═S or ═O.24. The compound of claim 23 wherein R¹ is --H or an optionallyprotected monosaccharide.
 25. The compound of claim 24 wherein theoptionally protected monosaccharide is 2'-deoxy-R²¹ -substituted ribose,wherein R²¹ is H, --OH, halogen or a moiety that enhances the nucleasestability of an oligonucleotide containing the optionally protected2'-deoxy-R²¹ -substituted ribose, 2'-deoxyribose or ribose.
 26. Thecompound of claim 25 wherein R²¹ is --H, --OH, --F, protected hydroxyl,--OCH₃, --O--CH₂ CH₃, --O--CH₂ CH₂ OH, --O--CH₂ CH₂ F, --O--CH₂ CH₂ CH₃,--O--CH₂ CH₂ CH₂ OH, --O--CH₂ CH₂ CH₂ F, --O--CH₂ CF₂ H, --O--CH₂ CF₃ or--O--CH₂ CH₂ --O--CH₃.
 27. The compound of claim 1 having the structure(1): ##STR51## and tautomers, solvates and salts thereof, wherein R¹ isa protecting group, an oligonucleotide, a nucleic acid, apolysaccharide, an optionally protected monosaccharide, hydroxyl,phosphate, hydrogen phosphonate, halo, azido, protected hydroxyl, or--H;R² is A(Z)_(X1), but R² is not amine, protected amine, nitro orcyano; R⁵ is H or a protecting group; R²⁷ is independently --CH═, --N═,--C(C₁ -C₈ alkyl)═ or --C(halogen)═, but no adjacent R²⁷ are both --N═,or two adjacent R²⁷ are taken together to form a ring having thestructure, ##STR52## R³⁴ is --O--, --S-- or --N(CH₃)--; R^(a) isindependently --CH═, --N═, --C(C₁₋₈ alkyl)═ or --C(halogen)═, but noadjacent R^(a) are both --N═; A is a backbone chain of 2-16 carbonatoms, any 1, 2 or 3 of which are optionally replaced with N, O or Satoms, wherein the backbone chain is optionally substitutedindependently with 1, 2 or 3 of the following: C₁ -C₈ alkyl, --OR⁵, ═O,--NO₂, --N₃, --COOR⁵, --N(R⁵)₂, or --CN groups, C₁ -C₈ alkyl substitutedwith --OH, ═O, --NO₂, --N₃, --COOR⁵, --N(R⁵)₂, or --CN groups, or any ofthe foregoing in which --CH₂ -- is replaced with --O--, --NH-- or --N(C₁-C₈ alkyl); X1 is 1, 2 or 3; Y is H, 2-hydroxypyridine,N-hydroxysuccinimide, p-nitrophenyl, acylimidazole, maleimide,trifluoroacetate, an imido, a sulfonate, an imine 1,2-cyclohexanedione,glyoxal or an alpha-halo ketone; and Z independently is --NH₂, --CHO,--SH, --CO₂ Y, OY.
 28. The compound of claim 27 wherein Z is bonded to adetectable label.
 29. The compound of claim 27 wherein R¹ is anoligonucleotide.
 30. The compound of claim 27 wherein R¹ is anoptionally protected monosaccharide.